EP3695224A1 - Biomarqueurs de pronostic et de progression d'une néphropathie chronique - Google Patents

Biomarqueurs de pronostic et de progression d'une néphropathie chronique

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Publication number
EP3695224A1
EP3695224A1 EP18867154.9A EP18867154A EP3695224A1 EP 3695224 A1 EP3695224 A1 EP 3695224A1 EP 18867154 A EP18867154 A EP 18867154A EP 3695224 A1 EP3695224 A1 EP 3695224A1
Authority
EP
European Patent Office
Prior art keywords
subject
protein
uniprot accession
sample
amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18867154.9A
Other languages
German (de)
English (en)
Other versions
EP3695224A4 (fr
Inventor
Jennifer Van Eyk
Qin Fu
Vidya VENKATRAMAN
Zongming Fu
Josef Coresh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cedars Sinai Medical Center
Johns Hopkins University
Original Assignee
Cedars Sinai Medical Center
Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cedars Sinai Medical Center, Johns Hopkins University filed Critical Cedars Sinai Medical Center
Publication of EP3695224A1 publication Critical patent/EP3695224A1/fr
Publication of EP3695224A4 publication Critical patent/EP3695224A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • CKD chronic kidney disease
  • kidney disease Many people are at risk of developing kidney disease. As such there is a need for methods for identification/discoveiy of protein biomarkers for chronic kidney disease (CKD) and for diagnosing and/or prognosing and/or predicting progression of and/or treating chronic kidney disease (CKD).
  • CKD chronic kidney disease
  • the present invention provides a method for prognosing chrome kidne disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Fructose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-lRNA(Tyr) deacylase 1 (UniProt Accession No. Q8TEA8), Nek- associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No, Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (UniProt Accession No. Q27J81), F-actin-capping protein subunit beta (UniProt Accession No.
  • the mass spectrometry is selected from the group consisting of SRM, MRM, PRM, DDA, DIA, LC-MS, LC-MS/MS, LC-SRM-MS, LC-MRM-MS, LC- PRM-MS, LC-DDA-MS, LC-DIA-MS, and combinations thereof.
  • the subject is human.
  • the sample is plasma.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney disease.
  • the reference sample is obtained from the subject before the subject is treated for chronic kidney disease.
  • the reference sample is from a subject that has been successfully treated for chronic kidney disease.
  • the method further comprises administering a treatment to the subject. In some embodiments, the method further comprises, administering a treatment to the subject; obtaining a post- treatment sample from the subject, detecting the at least one protein in the post-treatment sample from the subject according to the method; determining the amount of the protein in the post-treatment sample from the subject according to the method, and comparing the amount of the protein in the post-treatment sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the post- treatment sample from the subject relative to the amount of the protein in the reference sample is indicative of the efficacy of the treatment.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney disease.
  • the reference sample is obtained from the subject before the subject is treated for chronic kidney disease.
  • the reference sample is from a subject that has been successfully treated for chronic kidney disease.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject; ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Neural ceil adhesion molecule 1 (UniProt Accession No, P13591), Dermcidin (DCD) (UniProt Accession No. P81605), Thyroxine-binding globulin (SERPINA7) (UniProt Accession No. P05543), 6- phosphofructokinase muscle type (PFKM) (UniProt Accession No. P08237), BTP (Prostaglandin-H2 D-isom erase) (UniProt Accession No. P41222), Leukocyte immunoglobulin-like receptor subfamily A member 3 (LILRA3) (UniProt Accession No.
  • the mass spectrometry is selected from the group consisting of SRM, MRM, PRM, DDA, DIA, LC-MS, LC-MS/MS, LC-SRM-MS, LC-MRM-MS, LC- PRM-MS, LC-DDA-MS, LC-DIA-MS, and combinations thereof.
  • the subject is human.
  • the sample is plasma.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney disease.
  • the reference sample is obtained from the subject before the subject is treated for chronic kidney disease.
  • the reference sample is from a subject that has been successfully treated for chronic kidney disease.
  • the method further comprises administering a treatment to the subject. In some embodiments, the method further comprises administering a treatment to the subject; obtaining a post-treatment sample from the subject; detecting the at least one protein in the post-treatment sample from the subject according to the method; determining the amount of the protein in the post-treatment sample from the subject according to the method, and comparing the amount of the protein in the post-treatment sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the post- treatment sample from the subject relative to the amount of the protein in the reference sample is indicative of the efficacy of the treatment.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney disease. In some embodiments, the reference sample is obtained from the subject before the subject is treated for chronic kidney disease. In some embodiments, the reference sample is from a subject that has been successfully treated for chronic kidney disease.
  • FIG, 1 depicts in accordance with various embodiments of the invention, a schematic representation of design for CKD discover)' (and verification/validation). Comparison of cases and controls at time 0 (baseline) yields candidate prognostic markers while comparison of changes over time in cases yields candidate progression markers.
  • FIG. 2 depicts in accordance with various embodiments of the invention validation of AlAGl (alpha- 1 -acid glycoprotein 1) and APOC3 (apolipoprotein CIII) protein expression in 16 controls and 16 CKD at both TO and Tl time points using targeted MRM assays.
  • ApoCIII (peptide GWVTDGFSSLK) (SEQ ID NO: 1) and AlAGl (peptide SDWYTDWK) (SEQ ID NO: 2)
  • SIL Stable Isotope Labelled
  • FIG. 2 discloses sequence SDVVY (SEQ ID NO: 3) and sequence GWVTDGFSSL (SEQ ID NO: 4).
  • FIG. 3 depicts in accordance with various embodiments of the invention, a schematic representation of cohort design for CKD discover ⁇ - (and verification/validation). Comparison of cases and controls at time 0 (baseline) will yield candidate prognostic markers while comparison of changes over time will yield candidate progression markers. Filtration and or severity markers can be potentially obtained by comparison at Tl of cases and controls.
  • FIG. 4 depicts in accordance with various embodiments of the invention, Ingenuity pathway analysis of top candidate severity (progression) biomarkers (p ⁇ 0.05) found TGFbeta as primary pathway.
  • the term “comprising” or “comprises” is used in reference to compositions, methods, systems, articles of manufacture, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).
  • sample is used herein in its broadest sense.
  • biological sample as used herein denotes a sample taken or isolated from a biological organism.
  • a sample or biological sample may comprise a bodily fluid including blood, serum, plasma, tears, aqueous and vitreous humor, spinal fluid; a soluble fraction of a cell or tissue preparation, or media in which cells were grown; or membrane isolated or extracted from a cell or tissue; polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue, a ti ssue print, a fingerprint, skin or hair; fragments and derivatives thereof.
  • samples or biological samples include cheek swab; mucus; whole blood, blood, serum; plasma; urine; saliva, semen; lymph; fecal extract; sputum; other body fluid or biofluid; cell sample; and tissue sample etc.
  • the term also includes a mixture of the above-mentioned samples or biological samples.
  • sample also includes untreated or pretreated (or pre-processed) biological samples.
  • a sample or biological sample can comprise one or more cells from the subject.
  • Subject samples or biological samples usually comprise derivatives of blood products, including blood, plasma and serum.
  • the sample is a biological sample.
  • the sample is blood.
  • the sample is plasma.
  • the sample is blood, plasma, serum, or urine.
  • body fluid or “bodily fluids” are liquids originating from inside the bodies of organisms.
  • Bodi ly fluids include amni otic fluid, aqueous humour, vitreous humour, bile, blood (e.g., serum), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph and perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (e.g., nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), serous fluid, semen, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, and vomit.
  • blood e.g., serum
  • breast milk e.g., breast milk
  • cerebrospinal fluid ce
  • Extracellular bodily fluids include intravascular fluid (blood plasma), interstitial fluids, lymphatic fluid and transcellular fluid.
  • Biological sample also includes a mixture of the above-mentioned body fluids.
  • Biological samples may be untreated or pretreated (or pre-processed) biological samples.
  • Sample collection procedures and devices known in the art are suitable for use with various embodiment of the present invention.
  • sample collection procedures and devices include but are not limited to: phlebotomy tubes (e.g., a vacutainer blood/specimen collection device for collection and/or storage of the blood/specimen), dried blood spots, Microvette CB300 Capillary Collection Device (Sarstedt), HemaXis blood collection devices (microfluidic technology, Hemaxis), Volumetric Absorptive Microsampling (such as CE-IVD Mitra microsampling device for accurate dried blood sampling (Neoteryx), HemaSpotTM-HF Blood Collection Device, a tissue sample collection device; standard collection/storage device (e.g., a collection/storage device for collection and/or storage of a sample (e.g., blood, plasma, serum, urine, etc.); a dried blood spot sampling device.
  • VAMS 1M the Volumetric Absorptive Microsampling
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, wood chucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, "patient”, “individual” and “subject” are used interchangeably herein.
  • the subject is mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.
  • the methods described herein can be used to treat domesticated animals and/or pets.
  • the subject is mouse or mice.
  • the subject is human.
  • the subject is selected from the group consisting of a subject suspected of having kidney disease, a subject that has kidney disease, a subject diagnosed with kidney disease, a subject that has been treated for kidney disease, a subject that is being treated for kidney disease, and a subject that is at risk of developing kidney disease.
  • the subject is selected from the group consisting of a subject suspected of having chronic kidney disease, a subject that has chronic kidney disease, a subject diagnosed with chrome kidney disease, a subject that has been treated for chronic kidne - disease, a subject that is being treated for chronic kidney disease, and a subject that is at risk of developing chrome kidney disease.
  • the subject is selected from the group consisting of a subject suspected of having a disease, a subject that has a disease, a subject diagnosed with a disease, a subject that has been treated for a disease, a subject that is being treated for a disease, and a subject that is at risk of developing a disease.
  • mammal refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats, laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino aci d, but that function s in a manner similar to a natural ly occurring amino acid.
  • a protein refers to any of a class of nitrogenous organic compounds that comprise large molecules composed of one or more long chains of amino acids and are an essential part of all living organisms.
  • a protein may contain various modifications to the amino acid structure such as disulfide bond formation, phosphorylations and glycosylations.
  • a linear chain of amino acid residues may be called a "polypeptide,"
  • a protein contains at least one polypeptide. Short polypeptides, e.g., containing less than 20-30 residues, are sometimes referred to as "peptides.”
  • threshold refers to the magnitude or intensity that must be exceeded for a certain reaction, phenomenon, result, or condition to occur or be considered relevant. The relevance can depend on context, e.g., it may refer to a positive, reactive or statistically significant relevance.
  • condition biological state or healt state
  • condition biological state or healt state
  • WHO biological state or healt state
  • health is understood in the present invention as status of a subject that can be described by physical, mental or social criteria. It includes as well so-called “healthy” and “diseased” conditions, therefore it is not limited to the WHO definition of health as "a state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity " but includes disease and infirmity.
  • disease refers to an abnormal condition affecting the body of an organism.
  • disorder refers to a functional abnormality or disturbance.
  • disease or disorder are used interchangeably herein unless otherwise noted or clear given the context in which the term is used,
  • state of health includes at least one condition as defined herein. It may also include a plurality of different conditions. In some embodiments, the state of health is a healthy state. In some embodiments, the state of health is a diseased state.
  • the term "healthy state” or “normal state” means that the state of a subject (e.g., biological state or health state, etc.) is not abnormal or does not comprise a disease or disorder.
  • a "healthy subject” or "normal subject” is a subject that does not have a disease or disorder.
  • the terms “treat”, “treatment”, “treating”, or “amelioration” when used in reference to a disease, disorder or medical condition refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to reverse, alleviate, ameliorate, inhibit, lessen, slow down or stop the progression or severity of a symptom, a condition, a disease, or a disorder.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, a disease, or a disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease, disorder or medical condition is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Also, “treatment” may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition, disease, or disorder even if the treatment is ultimately unsuccessful.
  • Those in need of treatment include those already with the condition, disease, or disorder as well as those prone to have the condition, disease, or disorder or those in whom the condition, disease, or disorder is to be prevented.
  • Non-limiting examples of treatments or therapeutic treatments include pharmacological or biological therapies and/or interventional surgical treatments.
  • preventative treatment means maintaining or improving a healthy state or non-diseased state of a healthy subject or subject that does not have a disease.
  • preventative treatment or “health surveillance” also means to prevent or to slow the appearance of symptoms associated with a condition, disease, or disorder.
  • preventative treatment also means to prevent or slow a subject from obtaining a condition, disease, or disorder.
  • the treatments or kits may be provided as pharmaceutical compositions.
  • the pharmaceutical compositions may be formulated for deliveiy via any route of administration.
  • Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, via inhalation, oral, transmucosal, transdermal, parenteral, enteral, topical or local.
  • Parenteral refers to a route of administration that is generally associated with injection, including intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
  • the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
  • the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.
  • the pharmaceutical compositions can be in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions or emulsions. Methods for these administrations are known to one skilled in the art.
  • the pharmaceutical compositions are formulated for intravascular, intravenous, or intraarterial administration.
  • the pharmaceutical compositions can contain any pharmaceutically acceptable excipient.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, nontoxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • excipients include but are not limited to starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emu!sifiers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof
  • the pharmaceutical compositions can contain any pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
  • Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its imaging benefits.
  • the pharmaceutical compositions can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.
  • Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water.
  • Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • compositions are made following the conventional techniques of pharmacy involving dry milling, mixing, and blending for powder forms, milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
  • Such a liquid formulation may be administered directly p.o, or filled into a soft gelatin capsule.
  • formulants may be added to the pharmaceutical composition.
  • a liquid formulation may be preferred.
  • these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, bulking agents or combinations thereof.
  • Carbohydrate formulants include sugar or sugar alcohols such as monosaccharides, disaccharides, or polysaccharides, or water soluble glucans.
  • the saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, puliulan, dextrin, alpha and beta cyclodextnn, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof.
  • “Sugar alcohol” is defined as a C4 to C8 hydrocarbon having an -OH group and includes gaiactitol, inositol, mannitol, xylitoi, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. In one embodiment, the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, in some embodiments between 2.0 and 6.0 w/v %.
  • Polymers formulants include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000.
  • PVP polyvinylpyrrolidone
  • PEG polyethylene glycol
  • a buffer may also be used in the pharmaceutical compositions to minimize pH changes in the solution before lyophilization or after reconstitution.
  • Most any physiological buffer may be used including but not limited to citrate, phosphate, succinate, and giutamate buffers or mixtures thereof.
  • the concentration is from 0.01 to 0.3 molar.
  • Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,110.
  • the pharmaceutical composition may be lyophilized to prevent degradation and to preserve sterility.
  • Methods for lyophilizing pharmaceutical compositions are known to those of ordinary skill in the art.
  • the phamiaceutical composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients.
  • the pharmaceutical composition is administered to subjects using those methods that are known to those skilled in the art.
  • the pharmaceutical compositions may be sterilized by conventional, well-known sterilization techniques.
  • the resulting solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the pharmaceutical compositions may contain pharmaceutically-acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and stabilizers (e.g., 1-20% maltose, etc.).
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, time, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
  • “Beneficial results” or “desired results” may include, but are in no way limited to, lessening or alleviating the severity of the disease or condition, preventing the disease or condition from worsening, curing the disease or condition, preventing the disease or condition from developing, lowering the chances of a patient developing the disease or condition, decreasing morbidity and mortality, and prolonging a patient's life or life expectancy.
  • "beneficial results” or “desired results” may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of chronic kidney disease, delay or slowing of chronic kidney disease, and amelioration or palliation of symptoms associated with chronic kidney disease.
  • administering refers to the placement an agent or a treatment as disclosed herein into a subject by a method or route which results in at least partial localization of the agent or treatment at a desired site.
  • Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, via inhalation, oral, anal, intra-anal, peri-anal, transmucosal, transdermal, parenteral, enteral, topical or local.
  • Parenteral refers to a route of administration that is generally associated with injection, including intratumoral, intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemai, intrathecal, intrauterine, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
  • the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
  • the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.
  • the pharmaceutical compositions can be in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions or emulsions.
  • “administering” can be self-administering. For example, it is considered as “administering" that a subject consumes a composition as disclosed herein,
  • Diagnostic means identifying the presence or nature of a pathologic condition, disease, or disorder and includes identifying patients who are at risk of developing a specific condition, disease or disorder. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
  • the "specificity" of a diagnostic assay is J minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, a disease, or a disorder, it suffices if the method provides a positive indication that aids in diagnosis.
  • At risk of is intended to mean at increased risk of, compared to a normal subject, or compared to a control group, e.g. a patient population.
  • a subject carrying a particular marker may have an increased risk for a specific condition, disease or disorder, and be identified as needing further testing.
  • Increased risk or “elevated risk” mean any statistically significant increase in the probability, e.g., that the subject has the disorder. The risk is increased by at least 10%, at least 20%, and even at least 50% over the control group with which the comparison is being made.
  • statically significant refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p- value.
  • detection may be used in the context of detecting biomarkers, detecting peptides, detecting proteins, or of detecting a condition, detecting a disease or a disorder (e.g. when positive assay results are obtained). In the latter context, “detecting” and “diagnosing” are considered synonymous.
  • Antibody refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
  • the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)' 2 fragments.
  • the term "antibody,” as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHi, CH2 and CH3, but does not include the heavy- chain variable region.
  • Immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g., a marker).
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to i solate, target, and/or quantify the antigen.
  • Non-limiting examples of immunoassays include ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, SISCAPA (stable isotope standards and capture by anti-peptide antibodies), Western blot, etc.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition, disease, or disorder in need of treatment (e.g., chronic kidney disease) or one or more complications related to the condition, disease, or disorder, and optionally, have already undergone treatment for the condition, disease, disorder, or the one or more complications related to the condition, disease, or disorder.
  • a subject can al so be one who has not been previously diagnosed as having a condition, disease, or disorder or one or more complications related to the condition, disease, or disorder.
  • a subject can be one who exhibits one or more risk factors for a condition, disease, or disorder, or one or more complications related to the condition, disease, or disorder, or a subject who does not exhibit risk factors.
  • a "subject in need” of treatment for a particular condition, disease, or disorder can be a subject suspected of having that condition, disease, or disorder, diagnosed as having that condition, disease, or disorder, already treated or being treated for that condition, disease, or disorder, not treated for that condition, disease, or disorder, or at risk of developing that condition, disease, or disorder.
  • proteases and peptidases are used interchangeably herein to mean enzymes that breakdown proteins and peptides
  • phenotype as used herein comprises the composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior.
  • diagnosis refers to the identification of the nature and cause of a certain phenomenon.
  • a diagnosis typically refers to a medical diagnosis, which is the process of determining which disease or condition explains a symptoms and signs.
  • a diagnostic procedure often a diagnostic test or assay, can be used to provide a diagnosis.
  • a diagnosis can comprise detecting the presence of a disease or disorder, or condition.
  • prognosis refers to predicting the likely outcome of a current standing.
  • a prognosis can include the expected duration and course of a disease or disorder, such as progressive decline or expected recovery.
  • theranosis refers to a diagnosis or prognosis used in the context of a medical treatment.
  • theranostics can include diagnostic testing used for selecting appropriate and optimal therapies (or the inverse) based on the context of genetic content or other molecular or cellular analysis.
  • Theranostics includes pharmacogenomics, personalized and precision medicine.
  • “Beneficial results” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized ⁇ i.e., not worsening) state of progression, delay or slowing of progression or invasiveness, and amelioration or palliation of symptoms associated with the chronic kidney disease.
  • Treatment also includes a decrease in mortality or an increase in the lifespan of a subject as compared to one not receiving the treatment.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition, disease or disorder.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder described herein. Treatment is generally “effective” if one or more symptoms are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i .e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side- effects of the disease (including palliative treatment).
  • preventative treatment means maintaining or improving a healthy state or non-diseased state of a healthy subject or subject that does not have a disease.
  • preventative treatment also means to prevent or to slow the appearance of symptoms associated with a condition, disease, or disorder.
  • preventative treatment also means to prevent or slow a subject from obtaining a condition, disease, or disorder.
  • compositions and methods of the invention may be used to characterize a phenotype in a sample of interest.
  • the phenotype can be any phenotype of interest that may be characterized using the subject compositions and methods.
  • the characterizing may be providing a diagnosis, prognosis or theranosis for the disease or disorder.
  • a sample from a subject is analyzed using the compositions and methods of the invention. The analysis is then used to predict or determine the presence, stage, grade, outcome, or likely therapeutic response of a disease or disorder in the subject. The analysis can also be used to assist in making such prediction or determination.
  • the invention provides a method to identify protein biomarkers and patterns that are indicative of a disease. In various embodiments the invention provides a method to identify protein biomarkers and patterns that are indicative a disease is or may be present. In some embodiments these methods may provide objective rationale for further testing. In various embodiments the invention provides a method for the identification of a plurality of proteins from a sample, wherein each protein is correlated to one or more peptides, wherein each peptide is correlated to one or more transitions, wherein each transition comprises a Ql mass value.
  • the invention provides a method for the identification of a plurality of proteins from a sample, wherein each protein is correlated to one or more peptides, wherein each peptide is correlated to one or more transitions, wherein each transition comprises a Ql mass value and a Q3 mass value.
  • the invention provides a method for the identification of a plurality of proteins from a sample, wherein each protein is correlated to one or more peptides, wherein each peptide is correlated to one or more transitions, wherein each transition comprises a Q1/Q3 mass value pair.
  • SRM stands for selected reaction monitoring.
  • MRM stands for multiple reaction monitoring.
  • PRM stands for parallel reaction monitoring.
  • SWATH stands for sequential window acquisition of all theoretical fragment ion spectra.
  • DIA stands for data-independent acquisition.
  • MS stands for mass spectrometry.
  • SIL stands for stable isotope-labeled.
  • MS data can be raw MS data obtained from a mass spectrometer and/or processed MS data in which peptides and their fragments (e.g., transitions and MS peaks) are already identified, analyzed and/or quantified.
  • MS data can be Selective Reaction Monitoring (SRM) data, Multiple Reaction Monitoring (MRM) data, parallel reaction monitoring (PRM) data, Shotgun CID MS data, Original DIA MS Data, MSE MS data, p2CID MS Data, PAcIFIC MS Data, AIF MS Data, XDLA MS Data, SWATH MS data, or FT-ARM MS Data, or their combinations.
  • SRM Selective Reaction Monitoring
  • MRM Multiple Reaction Monitoring
  • PRM parallel reaction monitoring
  • SRM/MRM mass spectrometry is a technology with the potential for reliable and comprehensive quantification of substances of low abundance in complex samples.
  • SRM is performed on triple quadrupole- like instruments, in which increased selectivity is obtained through collision-induced dissociation. It is a non-scanning mass spectrometry technique, where two mass analyzers (Ql and Q3) are used as static mass filters, to monitor a particular fragment of a selected precursor.
  • Ql and Q3 two mass analyzers
  • various ionization methods can be used including without limitation electrospray ionization, chemical ionization, electron ionization, atmospheric pressure chemical ionization, and matrix-assisted laser desorption ionization.
  • Both the first mass analyzer and the collision cell are continuously exposed to ions from the source in a time dependent manner. Once the ions move into the third mass analyzer time dependence becomes a factor.
  • the first quadrapole mass filter, Ql is the primary m/z selector after the sample leaves the ionization source. Any ions with mass-to-charge ratios other than the one selected for will not be allowed to infiltrate Ql .
  • the collision cell, denoted as "q2", located between the first quadrapole mass filter Ql and second quadrapole mass filter Q3, is where fragmentation of the sample occurs in the presence of an inert gas like argon, helium, or nitrogen.
  • the fragmented ions Upon exiting the collision cell, the fragmented ions then travel onto the second quadrapole mass filter Q3, where m/z selection can occur again.
  • the specific pair of mass-over-charge (m/z) values associated to the precursor and fragment ions selected is referred to as a "transition".
  • the detector acts as a counting device for the ions matching the selected transition thereby returning an intensity distribution over time.
  • MRM is when multiple SRM transitions are measured within the same experiment on the chromatographic time scale by rapidly switching between the different precursor/fragment pairs.
  • the triple quadrupole instrument cycles through a series of transitions and records the signal of each transition as a function of the elution time. The method allows for additional selectivity by monitoring the chromatographic co-elution of multiple transitions for a given analyte.
  • PRM Parallel- Reaction Monitoring
  • PRM Parallel reaction monitoring
  • Ql selected peptide
  • PRM methodology uses the quadrupole of a mass spectrometer to isolate a target precursor ion, fragments the targeted precursor ion in the collision cell, and then detects the resulting product ions in the Orbitrap mass analyzer.
  • Quantification is carried out after data acquisition by extracting one or more fragment ions with 5-10 ppm mass windows.
  • PRM uses a quadrupole time-of-flight (QTOF) or hybrid quadrupole-orbitrap (QOrbitrap) mass spectrometer to carry out the peptides/ proteins quantitation.
  • QTOF include but are not limited to: TripleTOF® 6600 or 5600 System (Sciex); X500R QTOF System (Sciex); 6500 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) (Agilent); or Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometry (Waters).
  • QObitrap include but are not limited to: Q ExactiveTM Hybrid Quadrupole-Orbitrap Mass Spectrometer (the Thermo Scientific); or Orbitrap FusionTM TribridTM (the Thermo Scientific).
  • Non-limiting advantages of PRM include elimination of most interferences, provides more accuracy and attomole-level limits of detection and quantification, enables the confident confirmation of the peptide identity with spectral library matching, reduces assay development time since no target transitions need to be preselected, ensures UHPLC- compatible data acquisition speeds with spectrum multiplexing and advanced signal processing.
  • SWATH MS is a data independent acquisition (DIA) method which aims to complement traditional mass spectrometry-based proteomics techniques such as shotgun and SRM methods. In essence, it allows a complete and permanent recording of all fragment ions of the detectable peptide precursors present in a biological sample. It thus combines the advantages of shotgun (high throughput) with those of SRM (high reproducibility and consistency),
  • the developed methods herein can be applied to the quantification of polypeptides(s) or protein(s) in biological sample(s).
  • Any kind of biological samples comprising polypeptides or proteins can be the starting point and be analyzed by the methods herein.
  • any protein/peptide containing sample can be used for and analyzed by the methods produced here (e.g., tissues, cells).
  • the methods herein can also be used with peptide mixtures obtained by digestion. Digestion of a polypeptide or protein includes any- kind of cleavage strategies, such as, enzymatic, chemical, physical or combinations thereof.
  • the deciding factors of which polypeptide or protein will be the one of interest varies. It can be decided by performing a literature search and identifying proteins that are functionally related, are candidate protein biomarkers which can be used in screening for drug discovery, biomarker discovery and/or disease clinical phase trials or are diagnostic markers to screen for pharmaceutical/medical purposes.
  • the polypeptide or protein of interest may be determined by experimental analysis,
  • the following parameters of the methods provided herein are determined: trypsin (or other protease) digestion and peptide clean up, best responding polypeptides, best responding proteins, best responding peptides, best responding fragments, fragment intensity ratios (increased high and reproducible peak intensities), optimal collision energies, and all the optimal parameters to maximize sensitivity and/or specificity of the methods.
  • quantification of the polypeptides and/or of the corresponding proteins or activity/regulation of the corresponding proteins is desired.
  • a selected peptide is labeled with a stable-isotope and used as an internal standard to achieve absolute quantification of a protein of interest.
  • the analysis and/or comparison is done on protein samples of wild-type or physiological/healthy origin against protein samples of mutant or pathological origin,
  • the present invention supports the use of mass spectrometry as platform to identify signature polypeptides or proteins for quantitative proteomics.
  • the approach is applicable to the analysis of proteins from all organisms, from cells, organs, tissues, and in the context of in vivo and/or in vitro analyses.
  • Examples of applications of the invention include the development, use and commercialization of quantitative assays for sets of polypeptides or proteins of interest.
  • the invention can be beneficial for the pharmaceutical industry (e.g. drug development and assessment), the biotechnology industry (e.g. assay design and development and quality control), and in clinical applications (e.g. identification of biomarkers of disease and quantitative analysis for diagnostic, prognostic and/or therapeutic use).
  • the beta coefficients can be negative or positive, and have a t-value and significance of that t-value associated with each.
  • the t-value and significance assesses the extent to which the magnitude of the slope is significantly different from the line laying on the X-axis. If the beta coefficient is not statistically significant (i.e., the t-value is not significant), no statistical significance can be interpreted from that predictor. If the beta coefficient is significant, examine the sign of the beta. If the regression beta coefficient is positive, the interpretation is that for ever ⁇ ' l-unit increase in the predictor variable, the dependent variable will increase by the unstandardized beta coefficient value. For example, if the beta coefficient is .80 and statistically significant, then for each unit increase in the predictor variable, the outcome variable will increase by .80 units. The positive beta value indication of increased expression and negative beta value indicative of decreased expression.
  • marker or “biomarker” are used interchangeably herein, and in the context of the present invention refer to a protein or peptide (for example, protein or peptide associated with kidney disease or chronic kidney disease as described herein) is differentially present in a sample taken from patients having a specific disease or disorder as compared to a control value, the control value consisting of, for example average or mean values in comparable samples taken from control subjects (e.g., a person with a negative diagnosis, normal or healthy subject).
  • Biomarkers may be determined as specific peptides or proteins (Tables 1, 2, 6, and/or 7) which may be detected by antibodies or mass spectroscopy.
  • a mass spectroscopy or other profile of multiple antibodies may be used to determine multiple biomarkers, and differences between individual biomarkers and/or the partial or complete profile may be used for diagnosis.
  • the biomarkers may be detected by antibodies, mass spectrometry, or combinations thereof.
  • test amount of a marker refers to an amount of a marker present in a sample being tested.
  • a test amount can be either in absolute amount (e.g., g/mi) or a relative amount (e.g., relative intensity of signals).
  • a "diagnostic amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or disorder.
  • a diagnostic amount can be either in absolute amount (e.g., ⁇
  • a "control amount" of a marker can be any amount or a range of amount which is to be compared against a test amount of a marker.
  • a control amount of a marker can be the amount of a marker in a person who does not suffer from the disease or disorder sought to be diagnosed,
  • a control amount can be either in absolute amount (e.g., g/ml) or a relative amount (e.g., relative intensity of signals).
  • the term "differentially present” or “change in level” refers to differences in the quantity and/or the frequency of a marker present in a sample taken from patients having a specific disease or disorder as compared to a control subject.
  • a marker can be present at an elevated level or at a decreased level in samples of patients with the disease or disorder compared to a control value (e.g. determined from samples of control subjects).
  • a marker can be detected at a higher frequency or at a lower frequency in samples of patients compared to samples of control subjects.
  • a marker can be differentially present in terms of quantity, frequency or both as well as a ratio of differences between two or more specific modified amino acid residues and/or the enzyme itself.
  • an increase in the ratio of modified to unmodified proteins and peptides described herein is diagnostic of any one or more of the diseases described herein.
  • a marker, compound, composition or substance is differentially present in a sample if the amount of the marker, compound, composition or substance in the sample is statistically significantly different from the amount of the marker, compound, composition or substance in another sample, or from a control value.
  • a compound is differentially present if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%>, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater or less than it is present in the other sample (e.g. control), or if it is detectable in one sample and not detectable in the other.
  • a marker, compound, composition or substance is differentially present between samples if the frequency of detecting the marker, etc. in samples of patients suffering from a particular disease or disorder, is statistically significantly higher or lower than in the control samples or control values obtained from healthy individuals.
  • a biomarker is differentially present between the two sets of samples if it is detected at least about 0%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% more frequently or less frequently observed in one set of samples than the other set of samples.
  • Detectable moiety refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
  • the detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound detectable moiety in a sample.
  • the detectable moiety is a stable isotope.
  • the stable isotope is selected from the group consisting of i5 N, 13 C, 18 0, and 1.
  • binding assay is meant a biochemical assay wherein the biomarkers are detected by binding to an agent, such as an antibody, through which the detection process is carried out.
  • the detection process may involve radioactive or fluorescent labels, and the like.
  • the assay may involve immobilization of the biomarker, or may take place in solution.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • peptide refers to any compound containing at least two amino acid residues joined by an amide bond formed from the carboxyl group of one amino acid residue and the amino group of the adjacent amino acid residue.
  • peptide refers to a polymer of amino acid residues typically ranging in length from 2 to about 30, or to about 40, or to about 50, or to about 60, or to about 70 residues.
  • the peptide ranges in length from about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 60, 50, 45, 40, 45, 30, 25, 20, or 15 residues.
  • the peptide ranges in length from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25 residues.
  • the peptide ranges in length from 2 to about 12 residues, or 2 to about 20 residues, or 2 to about 30 residues, or 2 to about 40 residues, or 2 to about 50 residues, or 2 to about 60 residues, or 2 to about 70 residues.
  • the amino acid residues comprising the peptide are "L-form" amino acid residues, however, it is recognized that in various embodiments, "D" amino acids can be incorporated into the peptide.
  • Peptides also include amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the term applies to amino acids joined by a peptide linkage or by other, "modified linkages" (e.g., where the peptide bond is replaced by an a-ester, a f3-ester, a thioamide, phosphonamide, carbamate, hydroxylate, and the like (see, e.g., Spatola, (1983) Chern. Biochem. Amino Acids and Proteins 7: 267-357), where the amide is replaced with a saturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542, which is incorporated herein by reference, and Kaltenbronn eta/., (1990) Pp. 969-970 in Proc. ⁇ 1th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, and the like)).
  • modified linkages e.g., where the peptide bond is replaced by an a-ester, a f3-ester,
  • the disease referred to herein is kidney disease. In various embodiments, the disease referred to herein is chronic kidney disease,
  • the present invention provides a method for identifying protein biomarkers of chronic kidney disease in a subject, comprising: obtaining a sample from the subject, wherein the subject has chronic kidney disease, treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; detecting and/or measuring and/or quantifying the peptides in the digested sample, wherein the detecting and/or measuring and/or quantifying is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample; wherein protein biomarkers of chronic kidney disease are identified.
  • the detecting and/or measuring and/or quantifying the peptides in the digested sample is performed using an immunoassay or antibody method. In some embodiments, the detecting and/or measuring and/or quantifying the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • the measuring the peptides in the digested sample is performed using an immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for treating a subject in need thereof, comprising: obtaining protein biomarker signature results for the subject, wherein the protein biomarker signature results provide a prognosis of chronic kidney disease in the subject; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the prognosis of chronic kidney disease in the subject.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • the measuring the peptides in the digested sample is performed using immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof,
  • the present invention provides a method for treating a subject in need thereof, comprising: receiving protein biomarker signature results for the subject, wherein the protein biomarker signature results are indicative of progression of chronic kidney disease in the subject; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the progression of chronic kidney disease in the subject.
  • the present invention provides a method for diagnosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject, treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of chronic kidney disease in the subject.
  • the measuring the peptides in the digested sample is performed using immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for treating a subject in need thereof, comprising: receiving protein biomarker signature results for the subject, providing a diagnosis of chronic kidney disease in the subject based on the protein biomarker signature results; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the diagnosis,
  • the present invention provides a method for assessing and/or determining the risk of developing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides, measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, w herein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of an increased risk of the subject developing chronic kidney disease.
  • the measuring the peptides in the digested sample is performed using immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying and/or assessing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; comparing the protein biomarker signature from the subject to one or more reference protein biomarker signatures, and identifying and/or assessing chronic kidney disease in the subject based on the comparison.
  • the measuring the peptides in the digested sample is performed using immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying protein biomarkers of chronic kidney disease in a subject, comprising: obtaining a sample from the subject, wherein the subject has chronic kidney disease; detecting and/or measuring and/or quantifying one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the detecting and/or measuring and/or quantifying is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample; wherein protein biomarkers of chronic kidney disease are identified.
  • the detecting and/or measuring and/or quantifying the proteins in the sample is performed using an immunoassay or antibody method. In some embodiments, the detecting and/or measuring and/or quantifying the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying protein biomarkers of chronic kidney disease in a subject, comprising: obtaining a sample from the subject, wherein the subject has chronic kidney disease; detecting and/or measuring and/or quantifying one or more proteins in the sample, wherein the detecting and/or measuring and/or quantifying is performed using any one or more of an ELISA, immunoprecipitation, SISCAPA, Western blot, mass spectrometiv or combination thereof, wherein protein biomarkers of chronic kidney disease are identified.
  • the detecting and/or measuring and/or quantifying the proteins in the sample is performed using an immunoassay or antibody method.
  • the detecting and/or measuring and/or quantifying the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method. In some embodiments, measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucieic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the sample from the subject comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, or all of the proteins listed in Table 1.
  • the sample from the subject comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all of the proteins listed in Table 6.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: obtaining a sample from the subject, measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method.
  • measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the sample from the subject comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins listed in Table 2.
  • the sample from the subject comprises at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or all of the proteins listed in Table 7.
  • the present invention provides a method for diagnosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of chronic kidney- disease in the subject.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method.
  • measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for assessing and/or determining the risk of developing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of an increased risk of the subject developing chronic kidney disease.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method. In some embodiments, measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying and/or assessing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; comparing the protein biomarker signature from the subject to one or more reference protein biomarker signatures; and identifying and/or assessing chronic kidney disease in the subject based on the comparison.
  • measuring the proteins in the sample is performed using an antibody me immunoassay or antibody method.
  • the measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying protein biomarkers of chronic kidney disease in a subject, comprising: obtaining a sample from the subject, wherein the subject has chronic kidney disease, treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; detecting and/or measuring and/or quantifying the peptides in the digested sample, wherein the detecting and/or measuring and/or quantifying is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample; wherein protein biomarkers of chronic kidney disease are identified.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 2 and/or Table 6 and/or Table 7.
  • the subject is a human.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the mass spectrometry is multiple reaction monitoring (MRM).
  • MRM multiple reaction monitoring
  • the one or more proteins and/or one or more peptides in the sample are modified.
  • the one or more proteins and/or one or more peptides in the sample are chemically modified.
  • the modification is any one or more of any one or more of phosphorylation, methylation, acetylation, o-GlycNacylation, s-nitrosylation, citruilination, sumoylation, ubiquitinylation, neddylation, methyglyoxylation, or a post- translational modification.
  • the method further comprises adding one or more internal standards to the sample.
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof.
  • the reference sample is obtained from a control subject, wherein the control subject does not have the disease.
  • the detecting and/or measuring and/or quantifying the peptides in the digested sample is performed using an immunoassay or antibody method.
  • the detecting and/or measuring and/or quantifying the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 6.
  • the subject is a human.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase. carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the mass spectrometry is multiple reaction monitoring (MRM),
  • MRM multiple reaction monitoring
  • the one or more proteins and/or one or more peptides in the sample are modified.
  • the one or more proteins and/or one or more peptides in the sample are chemically modified.
  • the modification is any one or more of any one or more of phosphorylation, methylation, acetylation, o-GlycNacylation, s-nitrosylation, citrullination, sumoylation, ubiquitinylation, neddylation, methyglyoxylation, or a post-translational modification.
  • the method further comprises adding one or more internal standards to the sample.
  • the internal standard comprises one or more isotopicaliy labeled peptides, one or more isotopicaliy labeled proteins, or any combination thereof
  • the method further comprises treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the prognosis.
  • the method further comprises assessing the efficacy of the treatment, comprising comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of the efficacy of the treatment.
  • the reference sample is obtained from a control subject, wherein the control subject does not have the disease.
  • the reference sample is obtained from the subject before the subject is treated for the disease. In some embodiments, the reference sample is from a subject that has been successfully treated for the disease. In some embodiments, measuring the peptides in the digested sample is performed using an immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for treating a subject in need thereof, comprising: obtaining protein biomarker signature results for the subject, wherein the protein biomarker signature results provide a prognosis of chronic kidney- disease in the subject; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the prognosis of chronic kidney disease in the subject.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject, and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • the one or more peptides are correlated to one or more proteins according to Table 2 and/or Table 7.
  • the subject is a human.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase , subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxyiamine, or NTCB, or a combination thereof.
  • the mass spectrometry is multiple reaction monitoring (MRM).
  • the one or more proteins and/or one or more peptides in the sample are modified. In some embodiments, the one or more proteins and/or one or more peptides in the sample are chemically modified. In some embodiments, the modification is any one or more of any one or more of phosphorylation, methylation, acetylation, o-GlycNacylation, s-nitrosylation, citrullination, sumoylation, ubiquitinylation, neddylation, methyglyoxylation, or a post-translational modification. In some embodiments, the method further comprises adding one or more internal standards to the sample.
  • the internal standard comprises one or more isotopicaliy labeled peptides, one or more isotopicaliy labeled proteins, or any combination thereof
  • the method further comprises treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the progression of chronic kidney disease in the subject.
  • the method further comprises assessing the efficacy of the treatment, comprising comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of the efficacy of the treatment.
  • the reference sample is obtained from a control subject, wherein the control subject does not have the disease. In some embodiments, the reference sample is obtained from the subject before the subject is treated for the disease. In some embodiments, the reference sample is from a subject that has been successfully treated for the disease. In some embodiments, measuring the peptides in the digested sample is performed using an immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof,
  • the present invention provides a method for treating a subject in need thereof, comprising: receiving protein biomarker signature results for the subject, wherein the protein biomarker signature results are indicative of progression of chronic kidney disease in the subject; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the progression of chronic kidney disease in the subject.
  • the present invention provides a method for diagnosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject, treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of chronic kidney disease in the subject.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 2 and/or Table 6 and/or Table 7.
  • the subject is a human.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtil i sin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxyiamine, or NTCB, or a combination thereof.
  • the mass spectrometry is multiple reaction monitoring (MRM).
  • MRM multiple reaction monitoring
  • the one or more proteins and/or one or more peptides in the sample are modified.
  • the one or more proteins and/or one or more peptides in the sample are chemically modified.
  • the modification is any one or more of any one or more of phosphorylation, methylation, acetylation, o-GlycNacylation, s-nitrosylation, citmllination, sumoylation, ubiquitinylation, neddylation, methyglyoxylation, or a post-translational modification.
  • the method further comprises adding one or more internal standards to the sample.
  • the internal standard comprises one or more isotopicaliy labeled peptides, one or more isotopicaliy labeled proteins, or any combination thereof.
  • the method further comprises treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the diagnosis.
  • the method further comprises assessing the efficacy of the treatment, comprising comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of the efficacy of the treatment.
  • the reference sample is obtained from a control subject, wherein the control subject does not have the disease.
  • the reference sample is obtained from the subject before the subject is treated for the disease. In some embodiments, the reference sample is from a subject that has been successfully treated for the disease. In some embodiments, measuring the peptides in the digested sample is performed using an immunoassay or antibody method. In some embodiments, the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for treating a subject in need thereof, comprising: receiving protein biomarker signature results for the subject, providing a diagnosis of chronic kidney disease in the subject based on the protein biomarker signature results; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the diagnosis,
  • the present invention provides a method for assessing and/or determining the risk of developing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, w herein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of an increased risk of the subject developing chronic kidney disease.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 2 and/or Table 6 and/or Table 7.
  • measuring the peptides in the digested sample is performed using an immunoassay or antibody method.
  • the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, hi . I SA. immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying and/or assessing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring i s performed using any one or more of mass spectrometry, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; comparing the protein biomarker signature from the subject to one or more reference protein biomarker signatures; and identifying and/or assessing chronic kidney disease in the subject based on the comparison.
  • the one or more reference protein biomarker signatures are from one or more diseased subjects having chronic kidney disease. In some embodiments, the one or more reference protein biomarker signatures are from one or more healthy subjects. In some embodiments, the method further comprises treating the subject and/or selecting a treatment and/or providing a treatment and/or selecting a preventative treatment and/or providing a preventative treatment for the subject. In some embodiments, the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 2 and/or Table 6 and/or Table 7. In some embodiments, measuring the peptides in the digested sample is performed using an immunoassay or antibody method.
  • the measuring the peptides in the digested sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying protein biomarkers of chronic kidney disease in a subject, comprising: obtaining a sample from the subject, wherein the subject has chronic kidney disease; detecting and/or measuring and/or quantifying one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the detecting and/or measuring and/or quantifying is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample; wherein protein biomarkers of chronic kidney disease are identified.
  • the detecting and/or measuring and/or quantifying the proteins in the sample is performed using an immunoassay or antibody method. In some embodiments, the detecting and/or measuring and/or quantifying the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA,
  • the present invention provides a method for identifying protein biomarkers of chronic kidney disease in a subject, comprising: obtaining a sample from the subject, wherein the subject has chronic kidney disease; detecting and/or measuring and/or quantifying one or more proteins in the sample, wherein the detecting and/or measuring and/or quantifying is performed using any one or more of an ELISA, immunoprecipitation, SISCAPA, Western blot, mass spectrometiv or combination thereof, wherein protein biomarkers of chronic kidney disease are identified.
  • the detecting and/or measuring and/or quantifying the proteins in the sample is performed using an immunoassay or antibody method.
  • the detecting and/or measuring and/or quantifying the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method. In some embodiments, the measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method.
  • the measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for diagnosing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of chronic kidney- disease in the subject.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method.
  • the measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucieic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for assessing and/or determining the risk of developing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of an increased risk of the subject developing chronic kidney disease.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method. In some embodiments, the measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for identifying and/or assessing chronic kidney disease in a subject, comprising: obtaining a sample from the subject; measuring one or more proteins in the sample to obtain a protein biomarker signature for the subject, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; comparing the protein biomarker signature from the subject to one or more reference protein biomarker signatures, and identifying and/or assessing chronic kidney disease in the subject based on the comparison.
  • measuring the proteins in the sample is performed using an immunoassay or antibody method.
  • the measuring the proteins in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing and treating chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Fructose- bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No. P35555), CD59 glycoprotein (UniProt Accession No.
  • Apolipoprotein A-II (UniProt Accession No. P02652)
  • Pancreatic aipha-amylase (UniProt Accession No. P04746)
  • Coiled-coil domain-containing protein 80 (CCDC80) including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P8 J 605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Golgi membrane protein 1 (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No, Q8TEA8), Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (UniProt Accession No. Q27J81), F-actin-capping protein subu it beta (UniProt Accession No.
  • the determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing and treating chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combination thereof, wherein the protein is selected from the group consisting of Fructose-bisphosphateaidoiase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No.
  • CD59 glycoprotein (UniProt Accession No. P 13987), Apolipoprotein ⁇ - ⁇ (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No. P04746), Coiled-coil domain-containing protein 80 (CCDC80) including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P81605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Golgi membrane protein 1 (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No, Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No. Q8TEA8), Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (UniProt Accession No. Q27J81), F- actin-capping protein subunit beta (UniProt Accession No.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting and determining an amount of at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for determining progression of and treating chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Ql 5517), Isoform CI of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase-associated lipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No, Q14767), Neural cell adhesion molecule 1 (NCAM1 ) (UniProt Accession No.
  • iii) determining an amount of the protein in the sample from the subject; iv) constructing a protein biomarker signature for the subject, wherein the biomarker signature comprises the amount of the protein in the sample from the subject; v) comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is indicative of progression of chronic kidney disease in the subject; and vi) administering a treatment to the subject.
  • the determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for determining progression of and treating chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Q 15517). Isoform CI of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase- associated iipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q14767), Neural cell adhesion molecule 1 (NCAM1) (UniProt Accession No.
  • EMILIN-2 UniProt Accession No. Q9BXX0
  • combinations thereof iii) constructing a protein biomarker signature for the subject, wherein the biomarker signature comprises the amount of the protein in the sample from the subject; iv) comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is indicative of progression of chronic kidney disease in the subject, and v) administering a treatment to the subject.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometiy, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting and determining an amount of at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing and treating chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, wherein the sample comprises at least one protein, and wherein the protein is selected from the group consisting of Fructose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No. P35555), CD59 glycoprotein (UniProt Accession No. Pi 3987), Apolipoprotein ⁇ - ⁇ (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No.
  • Fructose-bisphosphatealdolase A UniProt Accession No. P04075
  • Fibrillin- 1 UniProt Accession No. P35555
  • CD59 glycoprotein UniProt Accession No. Pi 3987
  • Apolipoprotein ⁇ - ⁇ APOA2
  • Coiled-coil domain-containing protein 80 including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No, P02656), Dermcidin (UniProt Accession No. P81605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Golgi membrane protein 1 (UniProt Accession No. Q8NBJ4), T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No.
  • Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5 6), Inverted formin-2 (UniProt Accession No, Q27J81 ), F-actin-capping protein subunit beta (UniProt Accession No. P47756), Alpha-2-macroglobulin (UniProt Accession No. P01023), Leukocyte irnmunoglobulin-like receptor subfamily A member 3 (UniProt Accession No.
  • identifying the protein and determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCA A, Western blot, and combinations thereof.
  • the present invention provides a method for determining progression of and treating chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, wherein the sample comprises at least one protein, and wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium -coup led neutral amino acid transporter 10 (UniProt Accession No. Q9HBR0), Protein AMBP (precusors of alpha- 1 microglobulin) (UniProt Accession No. P02760), Aminopeptidase N (UniProt Accession No. PI 5144), C-reactive protein (CRP) (UniProt Accession No.
  • Uteroglobin UniProt Accession No. PI 1684
  • Putative sodium -coup led neutral amino acid transporter 10 UniProt Accession No. Q9HBR0
  • Protein AMBP precusors of alpha- 1 microglobulin
  • Vasodilator-stimulated phosphoprotein(VASP) (UniProt Accession No. P50552), Cystatin-C (UniProt Accession No. P01034), Calreticulin (UniProt Accession No. P27797), Caspase-14 (UniProt Accession No. P31944), Collagen aipha-2(I) chain (UniProt Accession No. P08123), Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No.
  • Isoform C I of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase-associated lipocalin (NGAL) (UniProt Accession No, P80188), Latent- transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule 1 (NCAMl) (UniProt Accession No. P13591), Dermcidin (DCD) (UniProt Accession No. P81605), Thyroxine-binding globulin (SERPINA7) (UniProt Accession No.
  • Q9BXX0 Q9BXX0
  • identifying the protein and determining an amount of the protein in the sample from the subject by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof, iii) constructing a protein biomarker signature for the subject, wherein the biomarker signature comprises the amount of the protein in the sample from the subject; and iv) comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is indicative of progression of chronic kidney disease in the subject; and v) administering a treatment to the subject.
  • identifying the protein and determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody- method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, wherein the sample comprises at least one protein, and wherein the protein is selected from the group consisting of Fructose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin-1 (UniProt Accession No. P35555), CD59 glycoprotein (UniProt Accession No. P13987), Apolipoprotein ⁇ - ⁇ (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No.
  • CCDC80 Coiled-coil domain-containing protein 80
  • CCDC80 Coiled-coil domain-containing protein 80
  • isoform 2 UniProt Accession No. Q76M96
  • Apolipoprotein C-III APOC3
  • Dermcidin UniProt Accession No. P81605
  • Polymeric immunoglobulin receptor UniProt Accession No. P01833
  • Goigi membrane protein I UniProt Accession No. Q8NBJ4
  • T-lymphoma invasion and metastasis-inducing protein 2 UniProt Accession No. Q8IVF5
  • D-tyrosyl-tRNA(Tyr) deacyiase 1 UniProt Accession No.
  • Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2 ⁇ associated protein (UniProt Accession No. Q9Y5K6), Inverted fomiin-2 (UniProt Accession No. Q27J81), F- actin-capping protein subunit beta (UniProt Accession No. P47756), Alpha-2-macroglobulin (UniProt Accession No. P01023), Leukocyte immunoglobulin-like receptor subfamily A member 3 (UniProt Accession No.
  • the mass spectrometry is selected from the group consisting of SRM, MRM, PRM, DDA, DIA, LC-MS, LC-MS/MS, LC-SRM-MS, LC-MRM-MS, LC-PRM-MS, LC-DDA-MS, LC-DIA-MS, and combinations thereof.
  • the subject is human.
  • the sample is plasma.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney disease.
  • the reference sample is obtained from the subject before the subject is treated for chronic kidney disease.
  • the reference sample is from a subject that has been successfully treated for chronic kidney disease.
  • the method further comprises administering a treatment to the subject. In some embodiments, the method further comprises administering a treatment to the subject; obtaining a post-treatment sample from the subject, wherein the post-treatment sample comprises at least one protein according to the method; identifying the protein in the post-treatment sample from the subject according to the method, determining an amount of the protein in the post-treatment sample from the subject according to the method; and comparing the amount of the protein in the post-treatment sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the post-treatment sample from the subject relative to the amount of the protein in the reference sample is indicative of the efficacy of the treatment.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney disease. In some embodiments, the reference sample is obtained from the subject before the subject is treated for chronic kidney disease. In some embodiments, the reference sample is from a subject that has been successfully treated for chronic kidney disease. In some embodiments, identifying the protein and determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof,
  • the sample from the subject comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins listed in Table 1.
  • the sample from the subject comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all of the proteins listed in Table 6.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, wherein the sample comprises at least one protein, and wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium- coupled neutral amino acid transporter 10 (UniProt Accession No. Q9HBR0), Protein AMBP (precusors of alpha- 1. microglobulin) (UniProt Accession No. P02760), Aminopeptidase N (UniProt Accession No. PI 5144), C-reactive protein (CRP) (UniProt Accession No.
  • Uteroglobin UniProt Accession No. PI 1684
  • Putative sodium- coupled neutral amino acid transporter 10 UniProt Accession No. Q9HBR0
  • Protein AMBP precusors of alpha- 1. microglobulin
  • Aminopeptidase N UniProt Acces
  • Vasodilator-stimulated phosphoprotein(VASP) (UniProt Accession No, P50552), Cystatin-C (UniProt Accession No, P01034), Calreticulin (UniProt Accession No. P27797), Caspase-14 (UniProt Accession No, P31944), Collagen alpha-2(I) chain (UniProt Accession No, P08123), Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No.
  • Isoform C I of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase- associated lipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural ceil adhesion molecule 1 (NCAM1) (UniProt Accession No. P13591), Dermcidin (DCD) (UniProt Accession No. P81605), Thyroxine-binding globulin (SERPINA7) (UniProt Accession No.
  • PFKM 6- phosphofructokinase muscle type
  • BTP Prostaglandin-H2 D-isom erase
  • LILRA3 Leukocyte immunoglobulin-like receptor subfamily A member 3
  • Cystatin-B UniProt Accession No, P04080
  • Plexin domain-containing protein 2 UniProt Accession No. Q6UX71
  • EMILIN-2 UniProt Accession No.
  • the mass spectrometry is selected from the group consisting of SRM, MRM, PRM, DDA, DIA, LC-MS, LC-MS/MS, LC-SRM-MS, LC-MRM- MS, LC-PRM-MS, LC-DDA-MS, LC-DIA-MS, and combinations thereof.
  • the subject is human.
  • the sample is plasma.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney disease.
  • the reference sample is obtained from the subject before the subject is treated for chronic kidney disease.
  • the reference sample is from a subject that has been successfully treated for chronic kidney disease.
  • the method further comprises administering a treatment to the subject. In some embodiments, the method further comprises administering a treatment to the subject; obtaining a post-treatment sample from the subject, wherein the post- treatment sample comprises at least one protein according to the method; identifying the protein in the post-treatment sample from the subject according to the method; determining an amount of the protein in the post-treatment sample from the subject according to the method; and comparing the amount of the protein in the post-treatment sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the post- treatment sample from the subject relative to the amount of the protein in the reference sample is indicative of the efficacy of the treatment.
  • the reference sample is obtained from a control subject, wherein the control subject does not have chronic kidney- disease. In some embodiments, the reference sample is obtained from the subject before the subject is treated for chronic kidney disease. In some embodiments, the reference sample is from a subject that has been successfully treated for chronic kidney disease. In some embodiments, identifying the protein and determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the sample from the subject comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins listed in Table 2.
  • the sample from the subject comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or all of the proteins listed in Table 7.
  • the present invention provides a method for prognosing chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Fructose- bisphosphatealdolase A (UniProt Accession No, P04075), Fibrillin- 1 (UniProt Accession No. P35555), CD59 glycoprotein (UniProt Accession No.
  • Apolipoprotein ⁇ - ⁇ (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No, P04746), Coiied-coil domain-containing protein 80 (CCDC80) including isoform 2 (UniProt Accession No, Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P8 605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Golgi membrane protein I (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-tRNA(Tyr) deacvlase 1 (LiniProt Accession No. Q8TEA8), Nck-associated protein 1 (UniProt Accession No, P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No, Q9Y5K6), Inverted formin-2 (UniProt Access! on No.
  • F-actin-capping protein sub unit beta (UniProt Accession No. P47756), Alpha-2-macroglobulin (UniProt Accession No, P01023), Leukocyte immunoglobulin-like receptor subfamily A member 3 (UniProt Accession No. Q8N6C8), Noelin including isoform 3 (UniProt Accession No. Q99784), Serum albumin (UniProt Accession No. P02768), Apolipoprotein LI (APOL1) (UniProt Accession No. 014791), Alpha- 1 -acid glycoprotein 1 (ORM1) (UniProt Accession No. P02763), Hornerin (UniProt Accession No.
  • the determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for prognosing chrome kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Fmctose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No.
  • CD59 glycoprotein (UniProt Accession No. P13987), Apolipoprotein A-H (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No. P04746), Coiled-coil domain-containing protein 80 (CCDC80) including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P81605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Goigi membrane protein 1 (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No. Q8TEA8), Nck- associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-assoeiated protein (UniProt Accession No. Q9Y5K6), Inverted forniin-2 (UniProt Accession No.
  • F-actin-capping protein subunit beta (UniProt Accession No. P47756), Alpha-2-macroglobulin (UniProt Accession No. P01023), Leukocyte immunoglobulin-iike receptor subfamily A member 3 (UniProt Accession No. Q8N6C8), Noelin including isoform 3 (UniProt Accession No. Q99784), Serum albumin (UniProt Accession No. P02768), Apolipoprotein LI (APOL l ) (UniProt Accession No. 014791), Alpha-l-acid glycoprotein I (ORMl) (UniProt Accession No. P02763), Hornerin (UniProt Accession No.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting and determining an amount of at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins are detected in the sample from the subject.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins are detected and/or an amount determined in the sample from the subject.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins from Table 1 are detected in the sample from the subject.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins from Table 1 are detected and/or an amount determined in the sample from the subject,
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all of the proteins from Table 6 are detected in the sample from the subject.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all of the proteins from Table 6 are detected and/or an amount determined in the sample from the subject.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Q15517), Isoform CI of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase-associated lipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule 1 (NCAM l) (UniProt Accession No.
  • EMILIN-2 UniProt Accession No. Q9BXX0
  • combinations thereof iii) determining an amount of the protein in the sample from the subject; iv) constructing a protein biomarker signature for the subject, wherein the biomarker signature comprises the amount of the protein in the sample from the subject; and v) comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is indicative of progression of chronic kidney disease in the subject.
  • the determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, hi . ISA. immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, irnmunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No, PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Q15517), Isoform C I of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase- associated iipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule 1 (NCAMl) (UniProt Accession No.
  • Plexin domain-containing protein 2 (UniProt Accession No, Q6UX71), EMILIN-2 (UniProt Accession No. Q9BXX0), and combinations thereof; iii) determining an amount of the protein in the sample from the subject; iv) constructing a protein biomarker signature for the subject, wherein the biomarker signature comprises the amount of the protein in the sample from the subject; and v) comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is indicative of progression of chronic kidney disease in the subject.
  • detecting at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the determining an amount of the protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • detecting and determining an amount of at least one protein in the sample is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or ail of the proteins are detected in the sample from the subject.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins are detected and/or determined in the sample from the subject.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all of the proteins from Table 2 are detected in the sample from the subject,
  • At least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, or all of the proteins from Table 2 are detected and/or determined in the sample from the subject.
  • At least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or ail of the proteins from Table 7 are detected in the sample from the subject,
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or all of the proteins from Table 7 are detected and/or determined in the sample from the subject.
  • the reference sample is a post-treatment reference sample.
  • the proteins in Table I are markers for prognosing chronic kidney disease. In some embodiments, the proteins in Table 6 are markers for prognosing chronic kidney disease.
  • the proteins in Table 2 are markers for determining progression of chronic kidney disease. In some embodiments, the proteins in Table 7 are markers for determining progression of chronic kidney disease.
  • an antibody method is selected from the group consisting of a polyclonal antibody method, monoclonal antibody method, synthetic antibody method, single antibody method, sandwich based antibody method, and combinations thereof.
  • an antibody method uses antibodies as capture reagents. In some embodiments, an antibody method uses antibodies as peptide capture reagents. In some embodiments, an antibody method uses antibodies as protein capture reagents.
  • a nucleic acid aptamer method uses nucleic acid aptamers as capture reagents. In some embodiments, a nucleic acid aptamer method uses nucleic acid aptamers as peptide capture reagents. In some embodiments, a nucleic acid aptamer method uses nucleic acid aptamers as protein capture reagents.
  • a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is an increase in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample.
  • a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is characterized by an increase in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample.
  • a difference in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is characterized by an increase in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample.
  • a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is a decrease in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample.
  • a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is characterized by a decrease in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample.
  • a difference in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is characterized by a decrease in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample.
  • comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample wherein a difference in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is a prognosis of chronic kidney disease in the subject.
  • comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample wherein a difference in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is indicative of progression of chronic kidney disease in the subject.
  • the present invention provides a method for prognosing chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject: ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCA A, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Fructose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No. Q8TEA8), Nek- associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-i (UniProt Accession No, Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (Uni rot Accession No. Q27J81), F-actin-capping protein subunit beta (UniProt Accession No.
  • the present invention provides a method for determining progression of chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject; ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No, P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Q 15517), Isoform CI of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase- associated lipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule 1 (NCAM1) (UniProt Accession No.
  • EMILIN-2 UniProt Accession No, Q9BXX0
  • combinations thereof iii) constructing a protein biomarker signature for the subject, wherein the biomarker signature comprises the amount of the protein in the sample from the subject; and iv) comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample, wherein a difference in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is indicative of progression of chronic kidney disease in the subject.
  • a method for prognosing chronic kidney disease in a subject comprising: i) obtaining a sample from the subject; ii) detecting the presence of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Fructose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No. P35555), CD59 glycoprotein (UniProt Accession No.
  • Apolipoprotein A-II (APOA2) (UniProt Accession No. P02652), Pancreatic alpha- amylase (UniProt Accession No. P04746), Coiled-coil domain-containing protein 80 (CCDC80) including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P81605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Golgi membrane protein 1 (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No. Q8TEA8), Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (UniProt Accession No. Q27J81), F-actin-capping protein subunit beta (UniProt Accession No.
  • a method for determining progression of chronic kidney disease in a subject comprising: i) obtaining a sample from the subject; ii) detecting the presence of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCA A, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No. Q9HBR0), Protein AMBP (precusors of alpha- 1.
  • microglobulin (UniProt Accession No. P02760), Aminopeptidase N (UniProt Accession No. PI 5144), C-reactive protein (CRP) (UniProt Accession No. P02741), Vasodilator-stimulated phosphoprotein(VASP) (UniProt Accession No. P50552), Cystatin-C (UniProt Accession No. P01034), Calreticulin (UniProt Accession No, P27797), Caspase- 14 (UniProt Accession No. P31944), Collagen alpha-2(I) chain (UniProt Accession No. P08123), Insulin-like growth factor-binding protein 6 (UniProt Accession No.
  • Neogenin (UniProt Accession No. Q92859), Corneodesmosm (UniProt Accession No. Q 15517), Isoform CI of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase-associated lipocalin (NGAL) (UniProt Accession No. P80188), Latent- transforming growth factor beta-binding protein 2 (UniProt Accession No. Q14767), Neural cell adhesion molecule 1 (NCAM1) (UniProt Accession No. P13591), Dermcidin (DCD) (UniProt Accession No.
  • NCAM1 Neural cell adhesion molecule 1
  • DCD Dermcidin
  • Thyroxine-binding globulin (UniProt Accession No. P05543), 6-phosphofructokinase muscle type (PFKM) (UniProt Accession No. P08237), BTP (Prostaglandin- ⁇ D-isomerase) (UniProt Accession No. P41222), Leukocyte immunoglobulin-like receptor subfamily A member 3 (LILRA3) (UniProt Accession No. Q8N6C8), Cystatin-B (UniProt Accession No. P04080), Plexin domain-containing protein 2 (UniProt Accession No. Q6UX71), EMILIN-2 (UniProt Accession No.
  • the mass spectrometer is a triple quadrupole mass spectrometer.
  • the mass spectrometer is a Triple-Time Of Flight (Triple- TOF) mass spectrometer configured for SWATH or a Q-Exactive mass spectrometer (Thermo Scientific), or any instrument with sufficiently high scan speed and a quadrupole mass filter to perform data independent acquisition.
  • Triple- TOF Triple-Time Of Flight
  • SWATH SWATH
  • Q-Exactive mass spectrometer Thermo Scientific
  • TQMS triple quadrupole mass spectrometers
  • Examples of triple quadrupole mass spectrometers (TQMS) that can perform MEM/SRM/SIM include but are not limited to: QTRAP ⁇ 6500 and 5500 System (Sciex); Triple QTripie Quad 6500 System (Sciex); Agilent 6400 Series Triple Quadrupole LC./MS systems; Thermo ScientificTM TSQTM Triple Quadrupole system; quadrupole time-of-flight (QTOF) mass spectrometers, or hybrid quadrupole-orbitrap (QOrbitrap) mass spectrometers to carry out the peptides/ proteins quantitation.
  • QTOF quadrupole time-of-flight
  • QOrbitrap hybrid quadrupole-orbitrap
  • quadrupole time-of-flight (QTOF) mass spectrometers include but are not limited to: TripleTOF® 6600 or 5600 System (Sciex); X500R QTOF System (Sciex); 6500 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) (Agilent); or Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometry (Waters).
  • hybrid quadrupole- orbitrap (QObitrap) mass spectrometers include but are not limited to: Q ExactiveTM Hybrid Quadrupole-Orbitrap Mass Spectrometer (the Thermo Scientific); or Orbitrap FusionTM TribridTM (the Thermo Scientific).
  • the mass spectrometry technique is tandem mass spectrometry (MS/MS).
  • the mass spectrometry technique is liquid chromatography-tandom mass spectrometry (LC-MS/MS).
  • the mass spectrometry technique is liquid chromatography-selected reaction monitoring-mass spectrometry (LC-SRM-MS).
  • the mass spectrometry technique is liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). In some embodiments, the mass spectrometry technique is selected reaction monitoring. In some embodiments, the mass spectrometry technique is multiple reaction monitoring (MRM). In some embodiments, the mass spectrometry technique is parallel reaction monitoring (PRM).
  • LC-MRM-MS liquid chromatography-multiple reaction monitoring-mass spectrometry
  • the mass spectrometry technique is selected reaction monitoring.
  • the mass spectrometry technique is multiple reaction monitoring (MRM).
  • the mass spectrometry technique is parallel reaction monitoring (PRM).
  • the mass spectrometry is selected from the group consisting of selected reaction monitoring (SRM), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data dependent acquisition (DDA), data independent acquisition (DIA), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandom mass spectrometry (LC-MS/MS), liquid chromatography-selected reaction monitoring-mass spectrometry (LC-SRM-MS), liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS), liquid chromatography-parallel reaction monitoring (LC-PRM- MS), liquid chromatography-data dependent acquisition-mass spectrometry (LC-DDA-MS), liquid chromatography-data independent acquisition-mass spectrometry (LC-DIA-MS), and combinations thereof.
  • SRM reaction monitoring
  • MRM multiple reaction monitoring
  • PRM parallel reaction monitoring
  • DDA data dependent acquisition
  • DIA data independent acquisition
  • LC-MS liquid chromatography-mass spectrometry
  • the samples are biological samples or complex biological samples.
  • the complex samples include, but are not limited to tissues and/or tissue extracts, and/or cells.
  • the peptides are derived by proteolysis or chemical cleavage of the polypeptide or protein.
  • a protease is utilized to cleave the polypeptide or protein into peptides.
  • the protease is trypsin.
  • proteases or cleavage agents may be used including but not limited to trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the protease is trypsin.
  • the proteins can be measured as intact mass using a mass spectrometer. In some embodiments, no digestion of the proteins is required prior to mass spectrometry,
  • the proteins can be measured as intact mass using an immunoassay. In some embodiments, no digestion of the proteins is required prior to performing an immunoassay.
  • the proteins can be measured as intact mass using a mass spectrometer, and immunoassay, or combination thereof. In some embodiments, no digestion of the proteins is required prior to mass spectrometry, immunoassay, or combination thereof.
  • the proteins can be measured as intact mass using antibody methods. In some embodiments, no digestion of the proteins is required prior to using the antibody methods.
  • the proteins can be measured as intact mass using nucleic acid aptamer methods. In some embodiments, no digestion of the proteins is required prior to using the nucleic acid aptamer methods.
  • a list of candidate peptides to be targeted for detection on the analytical instrument is generated by modeling protein cleavage.
  • a list of candidate peptides to be targeted for detection on the analytical instrument is generated by modeling trypsin digestion of the polypeptide or protein.
  • the list of candidate peptides is narrowed by eliminating peptides that, for example, cannot be detected on the analytical instrument.
  • a list of candidate peptides is narrowed by eliminating: a peptide that has not been previously detected on a mass spectrometer, a peptide susceptible to a modification that interferes with accurate quantitation, a miscleaved peptide comprising an internal protease recognition site, a peptide with relatively inaccessible ends evidenced by the presence of miscleaved peptides, a peptide that is not unique to the sequence of the protein of interest, a peptide not present in the mature protein, or a combination thereof.
  • the detection of a peptide is improved by changing the conditions for fragmenting that peptide prior to detecting a multiplicity of the peptides with the mass spectrometer.
  • the fragmentation condition is the collision energy.
  • the method further comprises adding a stable isotope-labeled peptide to the sample prior to mass spectrometry.
  • the absolute amount of a peptide in the sample is determined by comparing the MS signal s of natural and stable isotope-labeled peptides.
  • the comprehensive list of candidate peptides is narrowed by eliminating peptides.
  • conventional criteria are used to eliminate peptides from the comprehensive list of candidate peptides by eliminating peptides that: (i) were never detected by MS on any instrument, (ii) are not unique to the sequence of the protein of interest, (iii) are not located within the mature protein, (iv) contain amino acid residues such as methionine, cysteine, and/or asparagine that are subjected to posttranslational modifications that interfere with accurate quantitation by mass spectrometry, (v) are miscleaved or partially cleaved, (vi) are post-translationally modified in vivo, (vii) and/or a combination thereof.
  • transitions for each peptide with high and reproducible peak intensities are identified.
  • the collision energy for each transition is optimized.
  • mass spectrometry compri ses selected reaction monitoring (SRM), or multiple reaction monitoring (MRM).
  • SRM or MRM is performed on a triple quadrapole mass spectrometer.
  • the peptides uniquely associated with the polypeptide or protein of interest are those with high correlations, strong signals, high signal/noise and/or sequences unique to the protein of interest.
  • Selected-ion monitoring SRM or selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) provide the simplest method set up and the most selective and sensitive quantification.
  • SRM/MRM/SIM is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection.
  • TQMS triple quadrupole mass spectrometers
  • Sciex QTRAP® 6500 and 5500 System
  • Sciex Triple QTripie Quad 6500 System
  • Agilent 6400 Series Triple Quadrupole LC/MS systems or Thermo ScientificTM TSQTM Triple Quadrapole system .
  • stable i sotope-labeled peptide standards for absolute quantification are used.
  • the peptide labeled with a stable isotope is used as an internal standard to obtain absolute quantification of the polypeptide or protein of interest.
  • the peptides are quantified and then the amount of the parent protein present is inferred before digesting the sample with trypsin.
  • MS responses are used to determine an upper limit of quantification (ULOQ) and a lower limit of quantification (LLOQ).
  • the MS data comprises raw MS data obtained from a mass spectrometer and/or processed MS data in which peptides and their fragments (e.g., transitions and MS peaks) are already identified, analyzed and/or quantified.
  • the MS data is Selective Reaction Monitoring (SRM) data or Parallel-Reaction Monitoring (PRM) data and/or Multiple Reaction Monitoring (MRM) data.
  • the MS data is Shotgun CID MS data, Original DIA MS Data, MSE MS data, p2CID MS Data, PAcIFIC MS Data, AIF MS Data, XDLA MS Data, SWATH MS data, or I T-ARM MS Data, or a combination thereof.
  • acquiring MS data comprises operating a TripleTOF mass spectrometer, a triple quadrupole mass spectrometer, a liquid chromatography-mass spectrometry (LC-MS) system, a gas chromatography-mass spectrometry (GC-MS) system, or a tandem mass spectrometry (MS/MS) system, a dual time-of-flight (TOF-TOF) mass spectrometer, or a combination thereof.
  • LC-MS liquid chromatography-mass spectrometry
  • GC-MS gas chromatography-mass spectrometry
  • MS/MS tandem mass spectrometry
  • TOF-TOF dual time-of-flight
  • acquiring MS data comprises operating a mass spectrometer.
  • the mass spectrometer include but are not limited to high-resolution instruments such as Triple-TOF, Orbitrap, Fourier transform, and tandem time-of-flight (TOF/TOF) mass spectrometers: and high-sensitivity instruments such as triple quadrupole, ion trap, quadrupole TOF (QTOF), and Q trap mass spectrometers, and their hybrid and/or combination. High-resolution instruments are used to maximize the detection of peptides with minute mass-to-charge ratio (m/z) differences. Conversely, because targeted proteomics emphasize sensitivity and throughput, high-sensitivity instruments are used.
  • the mass spectrometer is a TripleTOF mass spectrometer.
  • the mass spectrometer is a triple quadrupole mass spectrometer.
  • the MS data is collected by a targeted acquisition method.
  • the targeted acquisition method include but are not limited to Selective Reaction Monitoring (SRM) and/or Multiple Reaction Monitoring (MRM) methods.
  • acquiring MS data comprises acquiring Selective Reaction Monitoring (SRM) data and/or Multiple Reaction Monitoring (MRM) data.
  • the MS data is collected by a data independent acquisition (DIA) method.
  • the MS data is collected by data dependent acquisition (DDA) method.
  • Non-limiting examples of mass spectrometry techniques include collision-induced dissociation (CD3), higher-energy coilisional dissociation (HCD), electron-transfer dissociation (ETD), etc,
  • MS Mass Spectrometry
  • LC-MS/MS liquid chromatography- tandem mass spectrometry
  • LC-SRM-MS liquid chromatography selected reaction monitoring mass spectrometry
  • CV% Coefficient of variation
  • SIL peptide Stable Isotope-Labeled Peptide
  • DIA-MS Data Independent acquisition mass spectrometry.
  • the kit is configured particularly for human subjects.
  • the kit is configured for research and/or veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals (e.g., mouse or mice).
  • the materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility.
  • the components can be in dissolved, dehydrated, or iyophiiized form; they can be provided at room, refrigerated or frozen temperatures.
  • the components are typically contained in suitable packaging material(s).
  • packaging material refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like.
  • the packaging material is constructed by well-known methods, to provide a sterile, contaminant-free environment.
  • the term "package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components.
  • the packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
  • the present invention provides a kit for identifying protein biomarkers of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof ; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to identify protein biomarkers of chronic kidney disease in the subject.
  • the kit further comprises instructions for using the kit to identify protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a kit for diagnosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation, and (d) instructions for using the kit to provide a diagnosis of chronic kidney disease in the subject.
  • the present invention provides a kit for prognosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases, (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to provide a prognosis of chronic kidney disease in the subject.
  • the present invention provides a kit for determining progression of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to determine progression of chronic kidney disease in the subject.
  • the present invention provides a kit for identifying protein biomarkers of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to identify protein biomarkers of chronic kidney disease in the subject.
  • the present invention provides a kit for diagnosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation, and (c) instructions for using the kit to provide a diagnosis of chronic kidney disease in the subject.
  • the present invention provides a kit for prognosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to provide a prognosis of chronic kidney disease in the subject,
  • the present invention provides a kit for determining progression of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to determine progression of chronic kidney disease in the subject.
  • the present invention provides a kit for identifying protein biomarkers of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to identify protein biomarkers of chronic kidney disease in the subject.
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase , subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxyiamine, or NTCB, or a combination thereof.
  • identification of protein biomarkers of chronic kidney disease comprises, obtaining a sample from the subject, wherein the subject has chronic kidney disease; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; detecting and/or measuring and/or quantifying the peptides in the digested sample, wherein the detecting and/or measuring and/or quantifying is performed using mass spectrometry; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the sample; and comparing the protein biomarker signature from the sample to a protein biomarker signature from a reference sample; wherein protein biomarkers of chronic kidney disease are identified.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 2 and/or Table 6 and/or Table 7.
  • the present invention provides a kit for identifying at least one protein biomarker of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to identify the protein biomarker of chronic kidney disease in the subject.
  • the internal standard comprises one or more proteins labeled with a detectable moiety, wherein the one or more proteins are selected from Table 1 and/or Table 2 and/or Table 6 and/or Table 7,
  • identification of the at least one protein biomarker of chronic kidney disease comprises, obtaining a sample from the subject, wherein the subject has chronic kidney disease; detecting and/or measuring and/or quantifying the protein biomarker in the sample, wherein the detecting and/or measuring and/or quantifying is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; constructing a protein biomarker signature for the subject from the sample; and comparing the protein biomarker signature from the subject's sample to a protein biomarker signature from a reference sample so as to identify protein biomarkers of chronic kidney disease in the
  • the present invention provides a kit for diagnosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases, (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to provide a diagnosis of chronic kidney disease in the subject.
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopicaliy labeled proteins, or any combination thereof.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxyl amine, or NTCB, or a combination thereof.
  • the diagnosis of chronic kidney disease comprises, obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a diagnosis of chronic kidney disease in the subject.
  • the one or more peptides are correlated to one or more proteins according to Table J and/or Table 2 and/or Table 6 and/or Table 7.
  • measuring the peptides in the digested sample is performed using at least one selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides a kit for diagnosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to provide a diagnosis of chronic kidney disease in the subject.
  • the internal standard comprises one or more proteins labeled with a detectable moiety, wherein the one or more proteins are selected from Table 1 and/or Table 2 and/or Table 6 and/or Table 7,
  • identification of the at least one protein biomarker of chronic kidney disease comprises, obtaining a sample from the subject; detecting and/or measuring and/or quantifying the protein biomarker in the sample, wherein the detecting and/or measuring and/or quantifying is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; constructing a protein biomarker signature for the subject from the sample, comparing the protein biomarker signature from the subject's sample to a protein biomarker signature from a reference sample; wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature
  • the present invention provides a kit for prognosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to provide a prognosis of chronic kidney disease in the subject.
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopicaliy labeled proteins, or any combination thereof.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermoiysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptida.se, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, orNTCB, or a combination thereof.
  • the prognosis of chronic kidney disease comprises, obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometn,', ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof, correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 6.
  • measuring the peptides in the digested sample is performed using at least one selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • present invention provides a kit for prognosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to provide a prognosis of chronic kidney- disease in the subject.
  • the internal standard comprises one or more proteins labeled with a detectable moiety, wherein the one or more proteins are selected from Table 1 and/or Table 6.
  • identification of the at least one protein biomarker of chronic kidney disease comprises, obtaining a sample from the subject; detecting and/or measuring and/or quantifying the protein biomarker in the sample, wherein the detecting and/or measuring and/or quantifying is performed using at least one technique selected from the group consisting of mass spectrometn,', antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; constructing a protein biomarker signature for the subject from the sample; comparing the protein biomarker signature from the subject's sample to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • the one or more protein biomarkers are selected from Table 1 and/or Table 6.
  • the present invention provides a kit for determining progression of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to determine progression of chronic kidney disease in the subject.
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermoiysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxyl amine, or NTCB, or a combination thereof.
  • determining the progression of chronic kidney disease comprises, obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • the one or more peptides are correlated to one or more proteins according to Table 2 and/or Table 7.
  • measuring the peptides in the digested sample is performed using at least one selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • present invention provides a kit for determining progression of chronic kidney disease in a subject, the kit compri sing: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to determine progression of chronic kidney disease in the subject.
  • the internal standard comprises one or more proteins labeled with a detectable moiety, wherein the one or more proteins are selected from Table 2 and/or Table 7.
  • identification of the at least one protein biomarker of chronic kidney disease comprises, obtaining a sample from the subject; detecting and/or measuring and/or quantifying the protein biomarker in the sample, wherein the detecting and/or measuring and/or quantifying is performed using at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, constructing a protein biomarker signature for the subject from the sample; comparing the protein biomarker signature from the subject's sample to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • the one or more protein biomarkers are selected from Table 2 and/or Table 7.
  • the present invention provides a kit for identifying protein biomarkers of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to identify protein biomarkers of chronic kidney disease in the subject.
  • the present invention provides a kit for diagnosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to provide a diagnosis of chronic kidney disease in the subject.
  • the present invention provides a kit for prognosing chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCA A, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to provide a prognosis of chronic kidney disease in the subject.
  • the present invention provides a kit for determining progression of chronic kidney disease in a subject, the kit comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) reagents and instructions for sample processing and preparation; and (c) instructions for using the kit to determine progression of chronic kidney disease in the subject.
  • the kit further comprises instructions for using the kit to identify protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the kit further comprises instructions for using the kit to identify protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, so as to provide a prognosis of chronic kidney disease in the subject.
  • the kit further comprises instructions for using the kit to identify protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, so as to determine progression of chronic kidney- disease in the subject.
  • the kit further comprises instructions for using the kit to identify and quantify protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the kit further comprises instructions for using the kit to identify and quantify protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, so as to provide a prognosis of chronic kidney disease in the subject.
  • the kit further comprises instructions for using the kit to identify and quantify protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISC APA, Western blot, and combinations thereof, so as to determine progression of chronic kidney disease in the subject.
  • a technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISC APA, Western blot, and combinations thereof, so as to determine progression of chronic kidney disease in the subject.
  • the kit further comprises instructions for using the kit to detect and determine an amount of protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof
  • the kit further comprises instructions for using the kit to detect and determine an amount of protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, so as to provide a prognosis of chronic kidney disease in the subject.
  • the kit further comprises instructions for using the kit to detect and determine an amount of protein biomarkers of chronic kidney disease by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, so as to determine progression of chronic kidney disease in the subject.
  • a technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, so as to determine progression of chronic kidney disease in the subject.
  • the present invention provides an assay for prognosing chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, wherein the sample comprises at least one protein, and wherein the protein is selected from the group consisting of Fructose-bisphosphateaidoiase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No. P35555), CD59 glycoprotein (UniProt Accession No. P 13987), Apolipoprotein ⁇ - ⁇ (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No.
  • Coiled-coil domain-containing protein 80 including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P81605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Golgi membrane protein I (UniProt Accession No. Q8NBJ4), T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No, Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No.
  • Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (UniProt Accession No. Q27J81), F- actin-capping protein subunit beta (UniProt Accession No. P47756), Alpha-2 ⁇ macroglobulin (UniProt Accession No. P01023), Leukocyte immunoglobulin-like receptor subfamily A member 3 (UniProt Accession No.
  • Q86YZ3 identifying the protein and determining an amount of the protein in the sample from the subject by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof, iii) constructing a protein biomarker signature for the subject, wherein the biomarker signature comprises the amount of the protein in the sample from the subject, and iv) comparing the amount of the protein in the sample from the subject to an amount of the protein in a reference sample, wherein a change in the amount of the protein in the sample from the subject relative to the amount of the protein in the reference sample is a prognosis of chronic kidney disease in the subject.
  • a technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, EL
  • the present invention provides an assay for determining progression of chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, wherein the sample comprises at least one protein, and wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. P I 1684), Putative sodium- coupled neutral amino acid transporter 10 (UniProt Accession No. Q9HBR0), Protein AMBP (precusors of alpha- 1 microglobulin) (UniProt Accession No. P02760), Aminopeptidase N (UniProt Accession No. PI 5144), C-reactive protein (CRP) (UniProt Accession No.
  • Uteroglobin UniProt Accession No. P I 1684
  • Putative sodium- coupled neutral amino acid transporter 10 UniProt Accession No. Q9HBR0
  • Protein AMBP precusors of alpha- 1 microglobulin
  • Aminopeptidase N UniProt Acces
  • Vasodilator-stimulated phosphoprotein(VASP) (UniProt Accession No, P50552), Cystatin-C (UniProt Accession No. P01034), Calreticulin (UniProt Accession No. P27797), Caspase-14 (UniProt Accession No, P31944), Collagen alpha ⁇ 2(I) chain (UniProt Accession No, PQ8123), Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No.
  • Isoform C I of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase- associated lipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule 1 (NCAMl) (UniProt Accession No, P13591), Dermcidin (DCD) (UniProt Accession No. P81605), Thyroxine-binding globulin (SERPINA7) (UniProt Accession No.
  • PFKM 6- phosphofructokinase muscle type
  • BTP Prostaglandin-H2 D-isom erase
  • LILRA3 Leukocyte immunoglobulin-like receptor subfamily A member 3
  • Cystatin-B UniProt Accession No, P04080
  • Plexin domain-containing protein 2 UniProt Accession No. Q6UX71
  • EMILIN-2 UniProt Accession No.
  • the present invention provides an assay for prognosing chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Fmctose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No.
  • CD59 glycoprotein (UniProt Accession No. P13987), Apolipoprotein A-II (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No. P04746), Coiled-coil domain-containing protein 80 (CCDC80) including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-III (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P81605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Golgi membrane protein 1 (UniProt Accession No.
  • T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No. Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No. Q8TEA8), Nck- associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (UniProt Accession No. Q27J81), F-actin-capping protein subunit beta (UniProt Accession No.
  • the determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides an assay for prognosing chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof: wherein the protein is selected from the group consisting of Fructose-bisphosphatealdolase A (UniProt Accession No. P04075), Fibrillin- ! (UniProt Accession No.
  • Golgi membrane protein 1 (UniProt Accession No, Q8NBJ4), T-lymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No, Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No, Q8TEA8), Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formiti-2 (UniProt Accession No.
  • F-actin-capping protein subunit beta (UniProt Accession No. P47756), Alpha-2-macroglobulin (UniProt Accession No. P01023), Leukocyte immunoglobulin-like receptor subfamily A member 3 (UniProt Accession No. Q8N6C8), Noelin including isoform 3 (UniProt Accession No. Q99784), Serum albumin (UniProt Accession No. P02768), Apolipoprotein LI (APOL1) (UniProt Accession No. 014791 ), Alpha- 1 -acid glycoprotein 1 (ORM1) (UniProt Accession No. P02763), Hornerin (UniProt Accession No.
  • the present invention provides an assay for determining progression of chronic kidney disease in a subject comprising: i) obtaining a sample from the subject, ii) detecting at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucieic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof: wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium -coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No, P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Q 15517), Isoform CI of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase- associated iipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule 1 (NCAM1) (UniProt Accession No.
  • the determining an amount of the protein in the sample from the subject is by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof.
  • the present invention provides an assay for determining progression of chronic kidney disease in a subject, comprising: i) obtaining a sample from the subject, ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, antibodies, nucleic acid aptamer method, nucleic acid aptamers, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Protein AMBP precusors of alpha- 1 microglobulin
  • Aminopeptidase N UniProt Accession No. PI 5144
  • C-reactive protein C-reactive protein
  • VASP Vasodilator-stimulated phosphoprotein
  • Cystatin-C UniProt Accession No. P01034
  • Calreticulin UniProt Accession No. P27797
  • Caspase-14 UniProt Accession No. P31944
  • Collagen alpha-2(I) chain UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No. P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Q15517), Isoform C I of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil geiatinase-associated lipocalin (NGAL) (UniProt Accession No. P80188), Latent- transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule I (NCAM1) (UniProt Accession No.
  • isoforms are determined based on peptide observations in the original discovery cohort
  • NGAL lipocalin
  • isoforms are determined based on peptide observations in the original discovery cohort
  • a method for identifying protein biomarkers of chronic kidney disease in a subject comprising: obtaining a sample from the subject, wherein the subject has chronic kidney- disease; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides, detecting and/or measuring and/or quantifying the peptides in the digested sample, wherein the detecting and/or measuring and/or quantifying is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample; wherein protein biomarkers of chronic kidney disease are identified.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 2 and/or Table 6 and/or Table 7.
  • protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermoiysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptida.se, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof.
  • kits for identifying protein biomarkers of chronic kidney disease in a subject comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to identify protein biomarkers of chronic kidney disease in the subject.
  • kits of paragraph 13 wherein the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof.
  • protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, sub tilt sin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxy! amine, or NTCB, or a combination thereof.
  • identification of protein biomarkers of chronic kidney disease comprises, obtaining a sample from the subject, wherein the subject has chronic kidney disease, treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; detecting and/or measuring and/or quantifying the peptides in the digested sample, wherein the detecting and/or measuring and/or quantifying is performed using mass spectrometry; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the sample; and comparing the protein biomarker signature from the sample to a protein biomarker signature from a reference sample, wherein protein biomarkers of chronic kidney disease are identified.
  • a method for prognosing chronic kidney disease in a subject comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 6.
  • protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof,
  • a method for assessing the efficacy of the treatment of claim 27, comprising: comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of the efficacy of the treatment.
  • a method for treating a subject in need thereof comprising: obtaining protein biomarker signature results for the subject, wherein the protein biomarker signature results provide a prognosis of chronic kidney disease in the subject; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the prognosis of chronic kidney disease in the subject.
  • a method for determining progression of chronic kidney disease in a subject comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISC APA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxy! amine, or NTCB, or a combination thereof.
  • the modification is any one or more of any one or more of phosphorylation, methylation, acetyiation, o-GlycNacylation, s-nitrosylation, citrullination, sumoylation, ubiquitinylation, neddylation, methyglyoxylation, or a post- translational modification.
  • a method for assessing the efficacy of the treatment of claim 43 comprising: comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of the efficacy of the treatment.
  • a method for treating a subject in need thereof comprising: receiving protein biomarker signature results for the subject, wherein the protein biomarker signature results are indicative of progression of chronic kidney disease in the subject: and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the progression of chronic kidney disease in the subject.
  • a method for diagnosing chronic kidney disease in a subject comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of chronic kidney disease in the subject.
  • protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxy peptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the modification is any one or more of any one or more of phosphorylation, methylation, acetylation, o-GlycNacylation, s-nitrosylation, citrullination, sumoylation, ubiquitinylation, neddylation, methyglyoxylation, or a post- transl ational modification.
  • a method for assessing the efficacy of the treatment of claim 59 comprising: comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of the efficacy of the treatment.
  • a method for treating a subject in need thereof comprising; receiving protein biomarker signature results for the subject, providing a diagnosis of chronic kidney disease in the subject based on the protein biomarker signature results; and treating the subject and/or selecting a treatment for and/or providing a treatment to the subject based on the diagnosis.
  • kits for diagnosing chronic kidney disease in a subject comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to provide a diagnosis of chronic kidney disease in the subject.
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the diagnosis of chronic kidney disease comprises, obtaining a sample from the subject, treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, w herein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a diagnosis of chronic kidney disease in the subject.
  • kit of paragraph 65 wherein the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 2 and/or Table 6 and/or Table 7.
  • kits for prognosing chronic kidney disease in a subject comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to provide a prognosis of chronic kidney disease in the subject.
  • the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, therm oly sin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the prognosis of chronic kidney disease comprises, obtaining a sample from the subject, treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, wherein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample provides a prognosis of chronic kidney disease in the subject.
  • kit of paragraph 70 wherein the one or more peptides are correlated to one or more proteins according to Table 1 and/or Table 6.
  • kits for determining progression of chronic kidney disease in a subject comprising: (a) one or more internal standards suitable for any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; (b) one or more proteases; (c) reagents and instructions for sample processing and preparation; and (d) instructions for using the kit to determine progression of chronic kidney disease in the subject,
  • kits of paragraph 77 wherein the internal standard comprises one or more isotopically labeled peptides, one or more isotopically labeled proteins, or any combination thereof.
  • protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • the protease is trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Asp-N, pepsin, thermolysin, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase, cathepsin C, cyanogen bromide, formic acid, hydroxylamine, or NTCB, or a combination thereof.
  • determining the progression of chronic kidney disease comprises, obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, w herein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of progression of chronic kidney disease in the subject.
  • a method for assessing and/or determining the risk of developing chronic kidney disease in a subject comprising: obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, ELIS A, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to a protein biomarker signature from a reference sample, w herein a change in the protein biomarker signature from the subject relative to the protein biomarker signature from the reference sample is indicative of an increased risk of the subject developing chronic kidney disease.
  • a method for identifying and/or assessing chronic kidney disease in a subject comprising; obtaining a sample from the subject; treating the sample with one or more proteases to obtain a digested sample comprising one or more peptides; measuring the peptides in the digested sample, wherein the measuring is performed using any one or more of mass spectrometry, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, or combination thereof; correlating the peptides to one or more proteins so as to obtain a protein biomarker signature for the subject; and comparing the protein biomarker signature from the subject to one or more reference protein biomarker signatures; and identifying and/or assessing chronic kidney disease in the subject based on the comparison,
  • a method for prognosing chronic kidney disease in a subject comprising: i) obtaining a sample from the subject; ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof, wherein the protein is selected from the group consisting of Fructose-bisphosphateaidoiase A (UniProt Accession No. P04075), Fibrillin- 1 (UniProt Accession No. P35555), CD59 glycoprotein (UniProt Accession No.
  • Apolipoprotein A-II (APOA2) (UniProt Accession No. P02652), Pancreatic alpha-amylase (UniProt Accession No. P04746), Coiled-coil domain-containing protein 80 (CCDC80) including isoform 2 (UniProt Accession No. Q76M96), Apolipoprotein C-0I (APOC3) (UniProt Accession No. P02656), Dermcidin (UniProt Accession No. P81605), Polymeric immunoglobulin receptor (UniProt Accession No. P01833), Goigi membrane protein 1 (UniProt Accession No.
  • T-Iymphoma invasion and metastasis-inducing protein 2 (UniProt Accession No, Q8IVF5), D-tyrosyl-tRNA(Tyr) deacylase 1 (UniProt Accession No. Q8TEA8), Nck-associated protein 1 (UniProt Accession No. P55160), Haptoglobin (UniProt Accession No. P00738), Talin-1 (UniProt Accession No. Q9Y490), CD2-associated protein (UniProt Accession No. Q9Y5K6), Inverted formin-2 (UniProt Accession No.
  • F- actin-capping protein subunit beta (UniProt Accession No, P47756), Alpha-2-macroglobulin (UniProt Accession No. P01023), Leukocyte immunoglobulin-like receptor subfamily A member 3 (UniProt Accession No. Q8N6C8), Noelin including isoform 3 (UniProt Accession No. Q99784), Serum albumin (UniProt Accession No. P02768), Apolipoprotein Ll (APOL l ) (UniProt Accession No. 014791), Alpha-l-acid glycoprotein I (ORMl) (UniProt Accession No, P02763), Hornerin (UniProt Accession No.
  • a method for determining progression of chronic kidney disease in a subject comprising: i) obtaining a sample from the subject; ii) detecting and determining an amount of at least one protein in the sample by at least one technique selected from the group consisting of mass spectrometry, antibody method, nucleic acid aptamer method, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, and combinations thereof; wherein the protein is selected from the group consisting of Uteroglobin (UniProt Accession No. PI 1684), Putative sodium-coupled neutral amino acid transporter 10 (UniProt Accession No.
  • Insulin-like growth factor-binding protein 6 (UniProt Accession No, P24592), Neogenin (UniProt Accession No. Q92859), Corneodesmosin (UniProt Accession No. Q 15517), Isoform CI of Tight junction protein ZO-2 (UniProt Accession No. Q9UDY2), Neutrophil gelatinase- associated iipocalin (NGAL) (UniProt Accession No. P80188), Latent-transforming growth factor beta-binding protein 2 (UniProt Accession No. Q 14767), Neural cell adhesion molecule 1 (NCAM1) (UniProt Accession No.
  • in-depth proteomics discovery is used to identify novel plasma biomarkers that are useful for the prognosis for development of chronic kidney disease (CKD) and to monitor progression (severity) of CKD. Furthermore, a subset of markers are related to key signaling pathways underlying the disease.
  • This example indicates that de novo proteomic analysis of plasma can be used to confirm changes of known biomarkers and to identify potential new biomarkers.
  • This example also shows that different plasma proteins may be aligned with prognosis versus severity.
  • proteins in the TGF beta pathway are particularly enriched as candidate severity biomarkers.
  • This study performs targeted verification and the validation of markers that have been identified in the de novo proteomics discovery phase.
  • a subset of top candidate markers are selected based on statistical and biological criteria and review for verification and the validation.
  • Biomarker development comprises various steps. These are discover ⁇ ', targeted assay validation (involving development and testing of the quantitative assays for each candidate proteins), verification and validation (carried out in various well phenotyped clinical cohorts). During each step, the number of candidate markers is reduced. After initial external validation, the number of biological samples (cohort size) is increased to test one or more biomarkers that can be used for diagnosis, prediction or as a target for therapy.
  • Table 3 and Table 1 and Table 2 list the candidate protein biomarkers for both prognosis and progression.
  • the selection of candidate biomarkers is based on statistical significance as well as knowledge about their biological and pathophysiological roles in CKD.
  • Our unbiased de novo discovery effort yielded interesting candidate biomarkers - among the very top hits listed in Table 3 are proteins whose blood levels were not previously known to be informative for CKD (APOL1 and fibrillin), a pathway (Complement - CD59 glycoprotein), and a protein (Haptoglobin).
  • Progression "top hits" are quite interesting with the top one (Uteroglobin) being stronger than cystatin and having low molecular weight and low missing rate.
  • the candidate proteins observed in the Discovery Phase using mass spectrometry are quantified by MRM.
  • ELISA is tested for specificity and sensitivity to ensure accuracy and agreement with initial discovery results (verification phase).
  • assays for verification and/or validation are selected from ELISA methods or MRM methods
  • LC-MS/MS analysis A Prominence UFLCXR HPLC system (Shimadzu) coupled to QTRAP® 6500 system (SCIEX) is used. Analyst software version 1 ,6.2 is applied to control the LC-MS/MS system and for data acquisition. Digested samples and N15 labeled peptides are separated on an Xbridge BEH30 C18 2.1mmxl00mm, 3.5 ⁇ column (Waters) maintained at 40°C. The mobile phase consists of A: 2% ACN, 98% water 0.1 % formic acid in water and B: 95% ACN (containing 0, 1 % formic acid) and the AB gradient is delivered at a flow rate of 0.25 ml/min. A two-phase switching valve is used for desalting and to divert the post-column eluent before entering the ion source of the MS instrument. [00275] Example 3.
  • Table 1 asid Table 2 list representatives of the top candidate proteins for prognosis and progression, respectively.
  • Quantitative assessment entails i) the selection of the top candidate biomarkers, ii) assessment of existing ELISA assay, iii) development of mass spectrometry (MS) based-multiple reaction monitoring (MRM) assays for the other top candidate biomarkers, iv) and the subsequent use of both ELISA and MRM assays for the quantification of these analytes in the two selected cohorts.
  • MS mass spectrometry
  • MRM multiple reaction monitoring
  • MRM mass spectrometry
  • Two or three unique peptides are measured and compared to a spiked synthetic peptide that has a i5 N amino acid (the labeled peptide is heavier than the endogenous peptide). Since the concentration of the l5 N labeled peptide is known, the quantity of the endogenous peptide, and thus, the concentration of the endogenous protein can be determined.
  • the rationale for defining cases by multiple criteria was done to increase specificity and ensured that we included the extreme cases based on the following rationale.
  • the rate of change of >-5 ml/min/year was chosen to be steeper than average for most CKD cohorts. This rate of change was determined by review of the progression rate in several CKD cohorts and clinical practice recommendations. Controls consisted of 16 individuals with stable GFR (decline ⁇ 1 ml/min/year for at least 3 years). Controls were matched on cohort based on age, sex, race, diabetes and hypertension. Both diabetics and non-diabetic participants in CRIC to ensure representation across this important risk factor of CKD.
  • the discover ⁇ ' pipeline consists of affinity depletion of the top 14 abundant plasma proteins, desalting and fractionation of intact low abundant proteins (flow through from the depletion column) using reversed phase HPLC into 2 fractions. Each fraction was neutralized, denatured, and digested with trypsin prior to analysis on the Orbitrap ELITE mass spectrometer (Thermo). Quality control was carried out at each step. Samples that deviated were rerun in their integrity. Each case and its matching control was blocked and run as group with baseline (TO) run prior to the subsequent sample (Tl), Samples within block were randomized, as were the 16 blocks, MS spectra was analyzed using OSMMA and X!
  • Tandem search engines on PASS platform Integrated Analysis. Isoforms must have at least one peptide to an amino acid sequence that is unique to a particular isoform. Uncharacterized or hypothesis proteins are researched using BLAST to ensure there is no common name. A total of 1551 non-redundant proteins (protein groups) were identified with a protein and peptide probability of ⁇ 0.1% with over 625,000 spectral counts.
  • a robot Biomek NX P that uses a 96 well plate format to carry out the core sample processing steps required for MRM. These include protein denaturation, reduction, alkylation and trypsin digestion. Testing of this system using standards protein and N 15 labeled peptides in plasma has shown that this step has %CV of less than 5% over many plates.
  • the desalting step which is required prior to MS analysis is traditionally carried out using reversed phase chromatography either in a pi pet tip or a 96 well format.
  • IP A Ingenuity Pathways Analysis
  • MRM Assays We perform MRM development, characterization, sample processing, running of samples and analysis. This lab uses either a 5500 or 6500 Q Trap with Turbo V Source with two or one XRMPX LC-20AD HPLC system (Shimadzu), that has flow rate of 100-200 uL/min with a normal run time of 15-30 min. This instalment configuration has exceptional stability, speed and potentially sensitivity that is required for clinical sample analysis. With this instrument we obtain excellent inter and intra-assay CV of ⁇ 2%, LLOD (which is peptide dependent) of 0.1 fmole and dynamic range of 10e6 to 10e7.
  • MRM assay development There are a number of steps involved in assay development. Briefly, the process of establishing an MRM assay consists of several steps: I) selection of peptide(s) unique to the protein of interest (prototypic peptides); 2) selection of the strongest fragment ion (each peptide parent-fragment pairing is called a MRM transition); 3) optimization for specific MS parameters (e.g. the collision energy) to maximize response and sensitivity; 4) multiplexing the peptide assays. Typically, we will use 2-3 peptides per target protein (and/or isoform) with peptide provides an independent quantitative value, a robustness not observed with western blots or ELISA.
  • MRM is linear over a concentration range of 1,000-fold and our current Triple Quadrupole MS instruments (5500 and 6500 Q-trap) have a percent coefficient of variance (%CV) of 2-5% for up to ' 1400 continuous runs (-2.5 weeks).
  • %CV percent coefficient of variance
  • MRMPilot IM Software to achieve automatic peptide and MRM transition selection from our MS/MS data as well as from in silico prediction.
  • MRMPilot also allows us automatic MRM method building and interactive optimized peptide MRM assay.
  • Multi Quant IM support relative and absolute quantification of peptides for candidates. This software allows us to handle large data sets (scheduled MRM algorithm and large number of patients).
  • Each MRM assays is tested in optimized in buffer to ensure maximum sensitivity, Standard curves are carried out in matrix to establish LLOD, LLOQ, ULOQ, ULOD and CV% for each assay.
  • Standard curves are carried out in matrix to establish LLOD, LLOQ, ULOQ, ULOD and CV% for each assay.
  • MRM Sample processing Throughput and reproducibility of sample preparation has improved with the development of robotic using 96 well plate taking 12 hours. Expectation is for this to be reduced to 2.5 hours.
  • Beta-gaiatosidase (a protein not found in blood) is added as a sample processing control.
  • MRM assays from Beta-galatosidase are included in all multiplexes. N l3 labeled peptides for each MRM assay is added at time of protein denaturation.
  • MRM analysis 10 point standard curves are run at the start and end of each batch, 3 point quality control standard curves are included 10% of the runs, each sample is run in triplicate. All sample with CV% greater than 20% (including sample preparation) are ream with appropriate standards. 35% of the data is manually inspected to ensure these is not baseline shift, each sample in that block is adjusted.

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Abstract

L'invention concerne des méthodes pour l'identification/la découverte de biomarqueurs protéiques d'une néphropathie chronique (CKD) et le diagnostic et/ou le pronostic et/ou la prédiction de la progression et/ou le traitement d'une néphropathie chronique (CKD).
EP18867154.9A 2017-10-12 2018-10-12 Biomarqueurs de pronostic et de progression d'une néphropathie chronique Withdrawn EP3695224A4 (fr)

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